Insect inhibitory proteins

ABSTRACT

A pesticidal protein class exhibiting toxic activity against Coleopteran and Lepidopteran pest species is disclosed, and includes, but is not limited to, TIC7040, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, and TIC7389. DNA constructs are provided which contain a recombinant nucleic acid sequence encoding the TIC7040, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, and TIC7389 pesticidal proteins. Transgenic plants, plant cells, seed, and plant parts resistant to Coleopteran and Lepidopteran infestation are provided which contain recombinant nucleic acid sequences encoding the TIC7040, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, and TIC7389 pesticidal proteins of the present invention. Methods for detecting the presence of the recombinant nucleic acid sequences or the proteins of the present invention in a biological sample, and methods of controlling Coleopteran and Lepidopteran species pests using the TIC7040, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, and TIC7389 pesticidal proteins are also provided.

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/480,614, filed Apr. 3, 2017, which is herein incorporated byreference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The file named “MONS443US-sequence_listing.txt” containing acomputer-readable form of the Sequence Listing was created on Apr. 2,2018. This file is 669,339 bytes (measured in MS-Windows®) iscontemporaneously filed by electronic submission (using the UnitedStates Patent Office EFS-Web filing system), and is incorporated intothis application by reference in its entirety.

FIELD OF THE INVENTION

The invention generally relates to the field of insect inhibitoryproteins. A novel class of proteins exhibiting insect inhibitoryactivity against agriculturally-relevant pests of crop plants and seedsis disclosed. In particular, the disclosed protein are insecticidallyactive against agriculturally-relevant pests of crop plants and seeds,particularly Coleopteran and Lepidopteran species of insect pests.Plants, plant parts, and seeds containing a recombinant polynucleotideconstruct encoding one or more of the disclosed toxin proteins areprovided.

BACKGROUND OF THE INVENTION

Improving crop yield from agriculturally significant plants including,among others, corn, soybean, sugarcane, rice, wheat, vegetables, andcotton, has become increasingly important. In addition to the growingneed for agricultural products to feed, clothe and provide energy for agrowing human population, climate-related effects and pressure from thegrowing population to use land other than for agricultural practices arepredicted to reduce the amount of arable land available for farming.These factors have led to grim forecasts of food security, particularlyin the absence of major improvements in plant biotechnology andagronomic practices. In light of these pressures, environmentallysustainable improvements in technology, agricultural techniques, andpest management are vital tools to expand crop production on the limitedamount of arable land available for farming.

Insects, particularly insects within the Lepidoptera and Coleopteraorders, are considered a major cause of damage to field crops, therebydecreasing crop yields over infested areas. Historically, the intensiveapplication of synthetic chemical insecticides was relied upon as thepest control agent in agriculture. Concerns for the environment andhuman health, in addition to emerging resistance issues, stimulated theresearch and development of biological pesticides. This research effortled to the progressive discovery and use of various entomopathogenicmicrobial species, including bacteria.

The biological control paradigm shifted when the potential ofentomopathogenic bacteria, especially bacteria belonging to the genusBacillus, was discovered and developed as a biological pest controlagent. Strains of the bacterium Bacillus thuringiensis (Bt) have beenused as a source for pesticidal proteins since it was discovered that Btstrains show a high toxicity against specific insects. Bt strains areknown to produce delta-endotoxins that are localized within parasporalcrystalline inclusion bodies at the onset of sporulation and during thestationary growth phase (e.g., Cry proteins), and are also known toproduce secreted insecticidal protein. Upon ingestion by a susceptibleinsect, delta-endotoxins as well as secreted toxins exert their effectsat the surface of the midgut epithelium, disrupting the cell membrane,leading to cell disruption and death. Genes encoding insecticidalproteins have also been identified in bacterial species other than Bt,including other Bacillus and a diversity of additional bacterialspecies, such as Brevibacillus laterosporus, Lysinibacillus sphaericus(“Ls” formerly known as Bacillus sphaericus) and Paenibacilluspopilliae.

Crystalline and secreted soluble insecticidal toxins are highly specificfor their hosts and have gained worldwide acceptance as alternatives tochemical insecticides. For example, insecticidal toxin proteins havebeen employed in various agricultural applications to protectagriculturally important plants from insect infestations, decrease theneed for chemical pesticide applications, and increase yields.Insecticidal toxin proteins are used to control agriculturally-relevantpests of crop plants by mechanical methods, such as spraying to dispersemicrobial formulations containing various bacteria strains onto plantsurfaces, and by using genetic transformation techniques to producetransgenic plants and seeds expressing insecticidal toxin protein.

The use of transgenic plants expressing insecticidal toxin proteins hasbeen globally adapted. For example, in 2012, 26.1 million hectares wereplanted with transgenic crops expressing Bt toxins (James, C., GlobalStatus of Commercialized Biotech/GM Crops: 2012. ISAAA Brief No. 44).The global use of transgenic insect-protected crops and the limitednumber of insecticidal toxin proteins used in these crops has created aselection pressure for existing insect alleles that impart resistance tothe currently-utilized insecticidal proteins.

The development of resistance in target pests to insecticidal toxinproteins creates the continuing need for discovery and development ofnew forms of insecticidal toxin proteins that are useful for managingthe increase in insect resistance to transgenic crops expressinginsecticidal toxin proteins. New protein toxins with improved efficacyand which exhibit control over a broader spectrum of susceptible insectspecies will reduce the number of surviving insects which can developresistance alleles. In addition, the use in one plant of two or moretransgenic insecticidal toxin proteins toxic to the same insect pest anddisplaying different modes of action reduces the probability ofresistance in any single target insect species.

Thus, the inventors herein disclose a novel protein toxin family fromBacillus laterosporous along with similar toxin proteins, variantproteins, and exemplary recombinant proteins that exhibit insecticidalactivity against target Lepidopteran and Coleopteran, particularlyagainst Western Corn Rootworm and Northern Corn Rootworm.

SUMMARY OF THE INVENTION

Disclosed herein is a novel group of pesticidal proteins with insectinhibitory activity (toxin proteins), referred to herein asTIC7040-related protein toxins, which are shown to exhibit inhibitoryactivity against one or more pests of crop plants. The TIC7040 proteinand proteins in the TIC7040 protein toxin class can be used alone or incombination with other insecticidal proteins and toxic agents informulations and in planta, thus providing alternatives to insecticidalproteins and insecticide chemistries currently in use in agriculturalsystems.

In one embodiment, disclosed in this application is a recombinantnucleic acid molecule comprising a heterologous promoter operably linkedto a polynucleotide segment encoding a pesticidal protein or fragmentthereof, wherein: (a) said pesticidal protein comprises the amino acidsequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, or SEQ IDNO:127; or (b) said pesticidal protein comprises an amino acid sequencehaving: (i) at least 75% identity to SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:75, SEQ ID NO:85, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ IDNO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ IDNO:107, or SEQ ID NO:109; or (ii) at least 80% to SEQ ID NO:111 SEQ IDNO: 113, SEQ ID NO:115, SEQ ID NO:119, SEQ ID NO:125, or SEQ ID NO:127;or (iii) at least 85% identity to SEQ ID NO:121 or SEQ ID NO:123; or(iv) at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:43, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:57, SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO:77, SEQ ID NO:79, SEQ NO:81, or SEQ ID NO:117; or (v)at least 93% identity to SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:30, andSEQ ID NO:59; or (vi) at least 94% identity to SEQ ID NO:53 and SEQ IDNO:55; or (vii) at least 99% identity to SEQ ID NO:32, SEQ ID NO:61, orSEQ ID NO:83; or (c) said polynucleotide segment hybridizes to apolynucleotide having the nucleotide sequence of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45,SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54,SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64,SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74,SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84,SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94,SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104,SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ IDNO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQID NO:124, OR SEQ ID NO:126; or (d) said recombinant nucleic acidmolecule is in operable linkage with a vector, and said vector isselected from the group consisting of a plasmid, phagemid, bacmid,cosmid, and a bacterial or yeast artificial chromosome. The recombinantnucleic acid molecule can comprise a sequence that functions to expressthe pesticidal protein in a plant; or is expressed in a plant cell toproduce a pesticidally effective amount of pesticidal protein.

In another embodiment of this application are host cells comprising arecombinant nucleic acid molecule of the application, wherein the hostcell is selected from the group consisting of a bacterial and a plantcell. Contemplated host cells include Agrobacterium, Rhizobium,Bacillus, Brevibacillus, Escherichia, Pseudomonas, Klebsiella, Pantoea,and Erwinia. In certain embodiments said Bacillus species is Bacilluscereus or Bacillus thuringiensis, said Brevibacillus is Brevibacilluslaterosperus, or said Escherichia is Escherichia coli. Contemplatedplant host cells include a dicotyledonous cell and a monocotyledonouscell. Further contemplated plant host cells include an alfalfa, banana,barley, bean, broccoli, cabbage, Brassica, carrot, cassava, castor,cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee,corn, clover, cotton (Gossypium sp.), a cucurbit, cucumber, Douglas fir,eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblollypine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasturegrass, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin,Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub,sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato,switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon,and wheat plant cell.

In yet another embodiment, the pesticidal protein exhibits activityagainst Coleopteran insect, including Western Corn Rootworm, SouthernCorn Rootworm, Northern Corn Rootworm, Mexican Corn Rootworm, BrazilianCorn Rootworm, Colorado Potato Beetle, Brazilian Corn Rootworm complexconsisting of Diabrotica viridula and Diabrotica speciosa, Crucifer FleaBeetle, Striped Flea Beetle, and Western Black Flea Beetle.

In another embodiment, the pesticidal protein exhibits activity againsta Lepidopteran insect, including Black Cutworm, Corn Earworm,Diamondback Moth, European Corn Borer, Fall Armyworm, Southern Armyworm,Soybean Looper, Southwestern Corn Borer, Tobacco Budworm, VelvetbeanCaterpillar, Sugarcane Borer, Lesser Cornstalk Borer, Black Armyworm,Beet Armyworm, Old World Bollworm, Oriental leaf Worm, or Pink Bollworm.

Also contemplated in this application are plants comprising arecombinant nucleic acid molecule comprising a heterologous promoteroperably linked to a polynucleotide segment encoding a pesticidalprotein or fragment thereof, wherein: (a) said pesticidal proteincomprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:43, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, or SEQ ID NO:127; or (b) said pesticidal proteincomprises an amino acid sequence having: (i) at least 75% identity toSEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26,SEQ ID NO:28, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69,SEQ ID NO:75, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91,SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101,SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, or SEQ ID NO:109; or (ii)at least 80% to SEQ ID NO:111 SEQ ID NO: 113, SEQ ID NO:115, SEQ IDNO:119, SEQ ID NO:125, or SEQ ID NO:127; or (iii) at least 85% identityto SEQ ID NO:121 or SEQ ID NO:123; or (iv) at least 90% identity to SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:14, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:57, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:77, SEQ IDNO:79, SEQ NO:81, or SEQ ID NO:117; or (v) at least 93% identity to SEQID NO:12, SEQ ID NO:16, SEQ ID NO:30, and SEQ ID NO:59; or (vi) at least94% identity to SEQ ID NO:53 and SEQ ID NO:55; or (vii) at least 99%identity to SEQ ID NO:32, SEQ ID NO:61, or SEQ ID NO:83; or (c) saidpolynucleotide segment hybridizes under stringent hybridizationconditions to the compliment of the nucleotide sequence of to SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ IDNO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ IDNO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ IDNO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120,SEQ ID NO:122, SEQ ID NO:124, OR SEQ ID NO:126; or (d) said plantexhibits a detectable amount of said pesticidal protein. In certainembodiments the pesticidal protein comprises SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:43, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, or SEQ ID NO:127. In one embodiment, the plant is eithera monocot or a dicot. In another embodiment, the plant is selected fromthe group consisting of an alfalfa, banana, barley, bean, broccoli,cabbage, Brassica, carrot, cassava, castor, cauliflower, celery,chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover,cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax,garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut,oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper,pigeon pea, pine, potato, poplar, pumpkin, Radiata pine, radish,rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southernpine, soybean, spinach, squash, strawberry, sugar beet, sugarcane,sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea,tobacco, tomato, triticale, turf grass, watermelon, and wheat.

In further embodiments, seeds comprising the recombinant nucleic acidmolecules are disclosed.

In another embodiment, an insect inhibitory composition comprising therecombinant nucleic acid molecules disclosed in this application arecontemplated. The insect inhibitory composition can further comprise anucleotide sequence encoding at least one other pesticidal agent that isdifferent from said pesticidal protein. The at least one otherpesticidal agent is selected from the group consisting of an insectinhibitory protein, an insect inhibitory dsRNA molecule, and anancillary protein. The at least one other pesticidal agent in the insectinhibitory composition exhibits activity against one or more pestspecies of the orders Lepidoptera, Coleoptera, or Hemiptera. The atleast one other pesticidal agent in the insect inhibitory composition isin one embodiment selected from the group consisting of: a Cry1A,Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1C, Cry1C variants, Cry1D,Cry1E, Cry1F, Cry1A/F chimeras, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K,Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variants, Cry3B, Cry4B, Cry6,Cry7, Cry8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B, Cry51Aa1, ET29,ET33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800,TIC807, TIC834, TIC853, TIC900, TIC901, TIC1201, TIC1415, TIC2160,TIC3131, TIC836, TIC860, TIC867, TIC869, TIC1100, VIP3A, VIP3B, VIP3Ab,AXMI-AXMI-, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100,AXMI-115, AXMI-113, and AXMI-005, AXMI134, AXMI-150, AXMI-171, AXMI-184,AXMI-196, AXMI-204, AXMI-207, AXMI-209, AXMI-205, AXMI-218, AXMI-220,AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z and AXMI-225z, AXMI-238,AXMI-270, AXMI-279, AXMI-345, AXMI-335, AXMI-R1 and variants thereof,IP3 and variants thereof, DIG-3, DIG-5, DIG-10, DIG-657 DIG-11,Cry71Aa1, Cry72Aa1, PHI-4 variants, PIP-72 variants, PIP-45 variants,PIP-64 variants, PIP-74 variants, PIP-75 variants, PIP-77 variants,Axmi422, Dig-305, Axmi440, PIP-47 variants, Axmi281, BT-009, BT-0012,BT-0013, BT-0023, BT0067, BT-0044, BT-0051, BT-0068, BT-0128, DIG-17,DIG-90, DIG-79, Cry1JP578V, Cry1JPS1, and Cry1 JPS1P578V.

Commodity products comprising a detectable amount of the recombinantnucleic acid molecules disclosed in this application are contemplated.Such commodity products include commodity corn bagged by a grainhandler, corn flakes, corn cakes, corn flour, corn meal, corn syrup,corn oil, corn silage, corn starch, corn cereal, and the like, andcorresponding cotton commodity products such as whole or processedcotton seed, cotton oil, lint, seeds and plant parts processed for feedor food, fiber, paper, biomasses, and fuel products such as fuel derivedfrom cotton oil or pellets derived from cotton gin waste, andcorresponding soybean commodity products such as whole or processedsoybean seed, soybean oil, soybean protein, soybean meal, soybean flour,soybean flakes, soybean bran, soybean milk, soybean cheese, soybeanwine, animal feed comprising soybean, paper comprising soybean, creamcomprising soybean, soybean biomass, and fuel products produced usingsoybean plants and soybean plant parts, and corresponding rice, wheat,sorghum, pigeon pea, peanut, fruit, melon, and vegetable commodityproducts including where applicable, juices, concentrates, jams,jellies, marmalades, and other edible forms of such commodity productscontaining a detectable amount of such polynucleotides and orpolypeptides of this application.

Also contemplated in this application is a method of producing seedcomprising the recombinant nucleic acid molecules disclosed in thisapplication. The method comprises planting at least one of the seedcomprising the recombinant nucleic acid molecules disclosed in thisapplication; growing plant from the seed; and harvesting seed from theplants, wherein the harvested seed comprises the recombinant nucleicacid molecules in this application.

In another illustrative embodiment, a plant resistant to insectinfestation is provided, wherein the cells of said plant comprise: (a) arecombinant nucleic acid molecule encoding an insecticidally effectiveamount of a pesticidal protein as set forth in SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:43, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, or SEQ ID NO:127; or (b) an insecticidally effectiveamount of a protein comprising an amino acid sequence having: (i) atleast 75% identity to SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:75, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, orSEQ ID NO:109; or (ii) at least 80% to SEQ ID NO:111 SEQ ID NO: 113, SEQID NO:115, SEQ ID NO:119, SEQ ID NO:125, or SEQ ID NO:127; or (iii) atleast 85% identity to SEQ ID NO:121 or SEQ ID NO:123; or (iv) at least90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:57, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:77, SEQ ID NO:79, SEQ NO:81, or SEQ ID NO:117; or (v) at least 93%identity to SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:30, and SEQ ID NO:59;or (vi) at least 94% identity to SEQ ID NO:53 and SEQ ID NO:55; or (vii)at least 99% identity to SEQ ID NO:32, SEQ ID NO:61, or SEQ ID NO:83.

Also disclosed in this application are methods for controlling aColeopteran or Lepidopteran species pest, and controlling a Coleopteranor Lepidopteran species pest infestation of a plant, particularly a cropplant. The method comprises, in one embodiment, (a) contacting the pestwith an insecticidally effective amount of one or more pesticidalproteins as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:43, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ IDNO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ IDNO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ IDNO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125,or SEQ ID NO:127; or (b) contacting the pest with an insecticidallyeffective amount of one or more pesticidal proteins comprising an aminoacid sequence having: (i) at least 75% identity to SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:75, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, or SEQ ID NO:109; or (ii) at least 80% to SEQ IDNO:111 SEQ ID NO: 113, SEQ ID NO:115, SEQ ID NO:119, SEQ ID NO:125, orSEQ ID NO:127; or (iii) at least 85% identity to SEQ ID NO:121 or SEQ IDNO:123; or (iv) at least 90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:34, SEQ IDNO:43, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:57, SEQ IDNO:71, SEQ ID NO:73, SEQ ID NO:77, SEQ ID NO:79, SEQ NO:81, or SEQ IDNO:117; or (v) at least 93% identity to SEQ ID NO:12, SEQ ID NO:16, SEQID NO:30, and SEQ ID NO:59; or (vi) at least 94% identity to SEQ IDNO:53 and SEQ ID NO:55; or (vii) at least 99% identity to SEQ ID NO:32,SEQ ID NO:61, or SEQ ID NO:83.

Further provided herein is a method of detecting the presence of arecombinant nucleic acid molecule comprising a polynucleotide segmentencoding a pesticidal protein or fragment thereof, wherein: (a) saidpesticidal protein comprises the amino acid sequence of SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ IDNO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ IDNO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ IDNO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121,SEQ ID NO:123, SEQ ID NO:125, or SEQ ID NO:127; or (b) said pesticidalprotein comprises an amino acid sequence having: (i) at least 75%identity to SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQID NO:26, SEQ ID NO:28, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:75, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ IDNO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, or SEQ ID NO:109;or (ii) at least 80% to SEQ ID NO:111 SEQ ID NO: 113, SEQ ID NO:115, SEQID NO:119, SEQ ID NO:125, or SEQ ID NO:127; or (iii) at least 85%identity to SEQ ID NO:121 or SEQ ID NO:123; or (iv) at least 90%identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:57, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:77, SEQ ID NO:79, SEQ NO:81, or SEQ ID NO:117; or (v) at least 93%identity to SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:30, and SEQ ID NO:59;or (vi) at least 94% identity to SEQ ID NO:53 and SEQ ID NO:55; or (vii)at least 99% identity to SEQ ID NO:32, SEQ ID NO:61, or SEQ ID NO:83; or(c) said polynucleotide segment hybridizes to a polynucleotide havingthe nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ IDNO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116,SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, or SEQ IDNO:126. In one embodiment of the invention, the method comprisescontacting a sample of nucleic acids with a nucleic acid probe thathybridizes under stringent hybridization conditions with genomic DNAfrom a plant comprising a polynucleotide segment encoding a pesticidalprotein or fragment thereof provided herein, and does not hybridizeunder such hybridization conditions with genomic DNA from an otherwiseisogenic plant that does not comprise the segment, wherein the probe ishomologous or complementary to SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60,SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:76,SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86,SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96,SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ IDNO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,OR SEQ ID NO:126, or a sequence that encodes a pesticidal proteincomprising an amino acid sequence having: (i) at least 75% identity toSEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26,SEQ ID NO:28, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69,SEQ ID NO:75, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91,SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101,SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, or SEQ ID NO:109; or (ii)at least 80% to SEQ ID NO:111 SEQ ID NO: 113, SEQ ID NO:115, SEQ IDNO:119, SEQ ID NO:125, or SEQ ID NO:127; or (iii) at least 85% identityto SEQ ID NO:121 or SEQ ID NO:123; or (iv) at least 90% identity to SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:14, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:57, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:77, SEQ IDNO:79, SEQ NO:81, or SEQ ID NO:117; or (v) at least 93% identity to SEQID NO:12, SEQ ID NO:16, SEQ ID NO:30, and SEQ ID NO:59; or (vi) at least94% identity to SEQ ID NO:53 and SEQ ID NO:55; or (vii) at least 99%identity to SEQ ID NO:32, SEQ ID NO:61, or SEQ ID NO:83. The method mayfurther comprise (a) subjecting the sample and probe to stringenthybridization conditions; and (b) detecting hybridization of the probewith DNA of the sample.

Also provided by the invention are methods of detecting the presence ofa pesticidal protein or fragment thereof in a sample comprising protein,wherein said pesticidal protein comprises the amino acid sequence of SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ IDNO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ IDNO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119,SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, or SEQ ID NO:127; or saidpesticidal protein comprises an amino acid sequence having: (i) at least75% identity to SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24,SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67,SEQ ID NO:69, SEQ ID NO:75, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89,SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99,SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, or SEQ IDNO:109; or (ii) at least 80% to SEQ ID NO:111 SEQ ID NO: 113, SEQ IDNO:115, SEQ ID NO:119, SEQ ID NO:125, or SEQ ID NO:127; or (iii) atleast 85% identity to SEQ ID NO:121 or SEQ ID NO:123; or (iv) at least90% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:14, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:57, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:77, SEQ ID NO:79, SEQ NO:81, or SEQ ID NO:117; or (v) at least 93%identity to SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:30, and SEQ ID NO:59;or (vi) at least 94% identity to SEQ ID NO:53 and SEQ ID NO:55; or (vii)at least 99% identity to SEQ ID NO:32, SEQ ID NO:61, or SEQ ID NO:83. Inone embodiment, the method comprises: (a) contacting a sample with animmunoreactive antibody; and (b) detecting the presence of the protein.In some embodiments the step of detecting comprises an ELISA, or aWestern blot.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a nucleic acid sequence obtained from Brevibacilluslaterosporus strain DSC005019 encoding a TIC7040 pesticidal proteinsequence.

SEQ ID NO:2 is the amino acid sequence of the TIC7040 protein.

SEQ ID NO:3 is a nucleic acid sequence obtained from Brevibacilluslaterosporus strain DSC005019 encoding a TIC7040HT pesticidal proteinsequence.

SEQ ID NO:4 is the amino acid sequence of the TIC7040HT protein.

SEQ ID NO:5 is a nucleic acid sequence encoding a TIC7040_4 pesticidalprotein sequence which comprises a C-terminal truncation relative to theTIC7040HT protein.

SEQ ID NO:6 is the amino acid sequence of the TIC7040_4 protein,consisting of amino acids 1 through 671 of TIC7040HT.

SEQ ID NO:7 is a nucleic acid sequence encoding a TIC7040_5 pesticidalprotein sequence which comprises an N-terminal and C-terminal truncationrelative to the TIC7040HT protein.

SEQ ID NO:8 is the amino acid sequence of the TIC7040_5 protein,comprising amino acids 13 through 611 of TIC7040HT.

SEQ ID NO:9 is a nucleic acid sequence encoding a TIC7040_6 pesticidalprotein sequence which comprises an N-terminal and C-terminal truncationrelative to the TIC7040HT protein.

SEQ ID NO:10 is the amino acid sequence of the TIC7040_6 protein,comprising amino acids 13 through 671 of TIC7040HT.

SEQ ID NO:11 is a nucleic acid sequence obtained from Brevibacilluslaterosporus strain DSC005973 encoding a TIC7042 pesticidal proteinsequence.

SEQ ID NO:12 is the amino acid sequence of the TIC7042 protein.

SEQ ID NO:13 is a nucleic acid sequence obtained from Brevibacilluslaterosporus strain DSC006713 encoding a TIC7381 pesticidal proteinsequence.

SEQ ID NO:14 is the amino acid sequence of the TIC7381 protein.

SEQ ID NO:15 is a nucleic acid sequence obtained from Brevibacilluslaterosporus strain DSC007657 encoding a TIC7382 pesticidal proteinsequence.

SEQ ID NO:16 is the amino acid sequence of the TIC7382 protein.

SEQ ID NO:17 is a nucleic acid sequence obtained from Brevibacilluslaterosporus strain DSC008106 encoding a TIC7383 pesticidal proteinsequence.

SEQ ID NO:18 is the amino acid sequence of the TIC7383 protein.

SEQ ID NO:19 is a nucleic acid sequence encoding a TIC7383_2 proteinwhich comprises an N-terminal truncation relative to the TIC7383protein.

SEQ ID NO:20 is the amino acid sequence of the TIC7383_2 protein,comprising amino acids 15 through 1256 of TIC7383.

SEQ ID NO:21 is a nucleic acid sequence encoding a TIC7383_3 proteinwhich comprises a C-terminal truncation relative to the TIC7383 protein.

SEQ ID NO:22 is the amino acid sequence of the TIC7383_3 protein andconsists of amino acids 1 through 659 of TIC7383.

SEQ ID NO:23 is a nucleic acid sequence encoding a TIC7383_4 proteinwhich comprises a C-terminal truncation relative to the TIC7383 protein.

SEQ ID NO:24 is the amino acid sequence of the TIC7383_4 protein andconsists of amino acids 1 through 679 of TIC7383.

SEQ ID NO:25 is a nucleic acid sequence encoding a TIC7383_5 proteinwhich comprises an N-terminal and C-terminal truncation relative to theTIC7383 protein.

SEQ ID NO:26 is the amino acid sequence of the TIC7383_5 protein andcomprises amino acids 15 through 659 of TIC7383.

SEQ ID NO:27 is a nucleic acid sequence encoding a TIC7383_6 proteinwhich comprises an N-terminal and C-terminal truncation relative to theTIC7383 protein.

SEQ ID NO:28 is the amino acid sequence of the TIC7383_6 protein,comprising amino acids 15 through 679 of TIC7383.

SEQ ID NO:29 is a nucleic acid sequence obtained from Brevibacilluslaterosporus strain DSC007651 encoding a TIC7386 pesticidal proteinsequence.

SEQ ID NO:30 is the amino acid sequence of the TIC7386 protein.

SEQ ID NO:31 is a nucleic acid sequence obtained from Brevibacilluslaterosporus strain DSC007962 encoding a TIC7388 pesticidal proteinsequence.

SEQ ID NO:32 is the amino acid sequence of the TIC7388 protein.

SEQ ID NO:33 is a nucleic acid sequence obtained from Brevibacilluslaterosporus strain DSC006878 encoding a TIC7389 pesticidal proteinsequence.

SEQ ID NO:34 is the amino acid sequence of the TIC7389 protein.

SEQ ID NO:35 is a synthetic coding sequence, CR-BRE1a.TIC7040.nno_Mc:1,encoding a TIC7040 pesticidal protein used for expression in plantcells.

SEQ ID NO:36 is a synthetic coding sequence,CR-BRE1a.TIC7040_10.nno_Mc:1, encoding a TIC7040HT pesticidal proteinused for expression in plant cells.

SEQ ID NO:37 is a synthetic coding sequence,CR-BRE1a.TIC7040_10.nno_Mc:3, encoding a TIC7040HT pesticidal proteinused for expression in plant cells.

SEQ ID NO:38 is a synthetic coding sequence,CR-BRE1a.TIC7040_10.nno_Mc:4, encoding a TIC7040HT pesticidal proteinused for expression in plant cells.

SEQ ID NO:39 is a synthetic coding sequence,CR-BRE1a.TIC7040_10.nno_Mc:5, encoding a TIC7040HT pesticidal proteinused for expression in plant cells.

SEQ ID NO:40 is a synthetic coding sequence,CR-BRE1a.TIC7040_10.nno_Mc:6, encoding a TIC7040HT pesticidal proteinused for expression in plant cells.

SEQ ID NO:41 is a synthetic coding sequence,CR-BRE1a.TIC7040_10.nno_Mc:7, encoding a TIC7040HT pesticidal proteinused for expression in plant cells.

SEQ ID NO:42 is a synthetic coding sequence,CR-BRE1a.TIC7040_1.nno_Mc:1, encoding a protein having an N-terminal andC-terminal truncation relative to the TIC7040HT protein used forexpression in plant cells.

SEQ ID NO:43 is the amino acid sequence of theCR-BRE1a.TIC7040_1.nno_Mc:1 protein, comprising amino acids 15 through651 of TIC7040HT.

SEQ ID NO: 44 is a synthetic coding sequence,CR-BRE1a.TIC7040_2.nno_Mc:1, encoding a TIC7040_6 (SEQ ID NO:10)pesticidal protein sequence which comprises an N-terminal and C-terminaltruncation relative to the TIC7040HT protein used for expression inplant cells.

SEQ ID NO:45 is a synthetic coding sequence,CR-BRE1a.TIC7040_11.nno_Mc:1, encoding a protein having an N-terminaland C-terminal truncation relative to the TIC7040HT protein used forexpression in plant cells.

SEQ ID NO:46 is the amino acid sequence of theCR-BRE1a.TIC7040_11.nno_Mc:1 protein, comprising amino acids 14 through671 of TIC7040HT.

SEQ ID NO:47 is a synthetic coding sequence,CR-BRE1a.TIC7040_12.nno_Mc:2, encoding a protein having a C-terminaltruncation relative to the TIC7040HT protein used for expression inplant cells.

SEQ ID NO:48 is the amino acid sequence of theCR-BRE1a.TIC7040_12.nno_Mc:2 protein, consisting of amino acids 1through 660 of TIC7040HT.

SEQ ID NO:49 is a synthetic coding sequence,CR-BRE1a.TIC7040_13.nno_Mc:1, which encodes a protein having aC-terminal truncation relative to the TIC7040HT protein used forexpression in plant cells.

SEQ ID NO:50 is the amino acid sequence of theCR-BRE1a.TIC7040_13.nno_Mc:1 protein, consisting of amino acids 1through 627 of TIC7040HT.

SEQ ID NO:51 is a synthetic coding sequence, CR-BRE1a.TIC7042.nno_Mc:1,which encodes a TIC7042 protein (SEQ ID NO:12) used for expression inplant cells.

SEQ ID NO:52 is a synthetic coding sequence,CR-BRE1a.TIC7042_1.nno_Mc:1, which encodes a protein having anN-terminal and C-terminal truncation relative to the TIC7042 proteinused for expression in plant cells.

SEQ ID NO:53 is the amino acid sequence of theCR-BRE1a.TIC7042_1.nno_Mc:1 protein, comprising amino acids 11 through646 of TIC7042.

SEQ ID NO:54 is a synthetic coding sequence,CR-BRE1a.TIC7042_2.nno_Mc:1, which encodes a protein having anN-terminal and C-terminal truncation relative to the TIC7042 proteinused for expression in plant cells.

SEQ ID NO:55 is the amino acid sequence of theCR-BRE1a.TIC7042_2.nno_Mc:1 protein, comprising amino acids 11 through665 of TIC7042.

SEQ ID NO:56 is a synthetic coding sequence,CR-BRE1a.TIC7381_1.nno_Mc:1, which encodes a TIC7381 protein wherein anadditional alanine codon is inserted immediately following theinitiating methionine codon used for expression in plant cells.

SEQ ID NO:57 is the amino acid sequence of CR-BRE1a.TIC7381_1.nno_Mc:1,wherein an additional alanine amino acid is inserted immediatelyfollowing the initiating methionine relative to the TIC7381 proteinsequence.

SEQ ID NO:58 is a synthetic coding sequence used for expression in plantcells, CR-BRE1a.TIC7382_1.nno_Mc:1, which encodes a TIC7382 proteinwherein an additional alanine codon is inserted immediately followingthe initiating methionine codon.

SEQ ID NO:59 is the amino acid sequence of CR-BRE1a.TIC7382_1.nno_Mc:1,wherein an additional alanine amino acid is inserted immediatelyfollowing the initiating methionine relative to the TIC7381 proteinsequence.

SEQ ID NO:60 is a synthetic coding sequence used for expression in plantcells, CR-BRE1a.TIC7382_2.nno_Mc:1, which encodes aCR-BRE1a.TIC7382_2.nno_Mc:1 protein comprising a C-terminal truncationrelative to the TIC7382 protein and wherein an additional alanine codonis inserted immediately following the initiating methionine codon.

SEQ ID NO:61 is the amino acid sequence of CR-BRE1a.TIC7382_2.nno_Mc:1comprising a C-terminal deletion and wherein an additional alanine aminoacid is inserted immediately following the initiating methioninerelative to the TIC7382 protein sequence, and comprises amino acids 1through 659 of TIC7382.

SEQ ID NO:62 is a synthetic coding sequence,CR-BRE1a.TIC7383_1.nno_Mc:1, which encodes a TIC7383 protein wherein anadditional alanine codon is inserted immediately following theinitiating methionine codon used for expression in plant cells.

SEQ ID NO:63 is the amino acid sequence of CR-BRE1a.TIC7383_1.nno_Mc:1,wherein an additional alanine amino acid is inserted immediatelyfollowing the initiating methionine relative to the TIC7383 proteinsequence.

SEQ ID NO:64 is a synthetic coding sequence,CR-BRE1a.TIC7383_7.nno_Mc:1, which encodes a CR-BRE1a.TIC7383_7.nno_Mc:1protein comprising an N-terminal and C-terminal truncation relative tothe TIC7383 protein and wherein an additional alanine codon is insertedimmediately following the initiating methionine codon and which is usedfor expression in plant cells.

SEQ ID NO:65 is the amino acid sequence of CR-BRE1a.TIC7383_7.nno_Mc:1comprising an N-terminal and C-terminal deletion and wherein anadditional alanine amino acid is inserted immediately following theinitiating methionine relative to the TIC7383 protein sequence, andcomprises amino acids 54 through 668 of TIC7383.

SEQ ID NO:66 is a synthetic coding sequence,CR-BRE1a.TIC7383_8.nno_Mc:1, which encodes a CR-BRE1a.TIC7383_8.nno_Mc:1protein comprising a C-terminal truncation relative to the TIC7383protein, wherein an additional alanine codon is inserted immediatelyfollowing the initiating methionine codon and which is used forexpression in plant cells.

SEQ ID NO:67 is the amino acid sequence of CR-BRE1a.TIC7383_8.nno_Mc:1comprising a C-terminal deletion, and wherein an additional alanineamino acid is inserted immediately following the initiating methioninerelative to the TIC7383 protein sequence, and comprises amino acids 1through 661 of TIC7383.

SEQ ID NO:68 is a synthetic coding sequence,CR-BRE1a.TIC7383_9.nno_Mc:1, which encodes a CR-BRE1a.TIC7383_9.nno_Mc:1protein comprising a C-terminal truncation relative to the TIC7383protein, wherein an additional alanine codon is inserted immediatelyfollowing the initiating methionine codon, and which is used forexpression in plant cells.

SEQ ID NO:69 is the amino acid sequence of CR-BRE1a.TIC7383_9.nno_Mc:1,comprising a C-terminal deletion wherein an additional alanine aminoacid is inserted immediately following the initiating methioninerelative to the TIC7383 protein sequence, and comprises amino acids 1through 668 of TIC7383.

SEQ ID NO:70 is a coding sequence encoding the tryptic core(TIC7040HT_Tryp) of the TIC7040HT protein as determined by massspectrometry.

SEQ ID NO:71 is the amino acid sequence of the tryptic core(TIC7040HT_Tryp) of the TIC7040HT protein as determined by massspectrometry, and comprises amino acids 43 through 624 of TIC7040HT.

SEQ ID NO:72 is a coding sequence encoding the chymotryptic core(TIC7040HT_Chymo) of the TIC7040HT protein as determined by massspectrometry.

SEQ ID NO:73 is the amino acid sequence of the chymotryptic core(TIC7040HT_Chymo) of the TIC7040HT protein as determined by massspectrometry, and comprises amino acids 45 through 641 of TIC7040HT.

SEQ ID NO:74 is a coding sequence encoding the tryptic core(TIC7383_Tryp) of the TIC7383 protein as determined by massspectrometry.

SEQ ID NO:75 is the amino acid sequence of the tryptic core(TIC7383_Tryp) of the TIC7383 protein as determined by massspectrometry, and comprises amino acids 55 through 668 of TIC7383.

SEQ ID NO:76 is a synthetic coding sequence,CR-BRE1a.TIC7040_14.nno_Mc:1, encoding a CR-BRE1a.TIC7040_14.nno_Mc:1protein having an N-terminal and C-terminal truncation relative to theTIC7040HT protein, wherein an additional alanine codon is insertedimmediately following the initiating methionine codon, and which is usedfor expression in plant cells.

SEQ ID NO:77 is the amino acid sequence of theCR-BRE1a.TIC7040_14.nno_Mc:1 protein, wherein an additional alanineamino acid is inserted immediately following the initiating methionine,and comprises amino acids 52 through 660 of TIC7040HT.

SEQ ID NO:78 is a synthetic coding sequence,CR-BRE1a.TIC7381_2.nno_Mc:1, encoding a CR-BRE1a.TIC7381_2.nno_Mc:1protein having a C-terminal truncation relative to the TIC7381 protein,wherein an additional alanine codon is inserted immediately followingthe initiating methionine codon, and which is used for expression inplant cells.

SEQ ID NO:79 is the amino acid sequence of theCR-BRE1a.TIC7381_2.nno_Mc:1 protein, wherein an additional alanine aminoacid is inserted immediately following the initiating methionine, andcomprises amino acids 1 through 658 of TIC7381.

SEQ ID NO:80 is a synthetic coding sequence,CR-BRE1a.TIC7381_3.nno_Mc:1, encoding a CR-BRE1a.TIC7381_3.nno_Mc:1protein having an N-terminal and C-terminal truncation relative to theTIC7381 protein, wherein an additional alanine codon is insertedimmediately following the initiating methionine codon, and which is usedfor expression in plant cells.

SEQ ID NO:81 is the amino acid sequence of theCR-BRE1a.TIC7381_3.nno_Mc:1 protein, wherein an additional alanine aminoacid is inserted immediately following the initiating methionine, andcomprises amino acids 50 through 658 of TICTIC7381.

SEQ ID NO:82 is a synthetic coding sequence,CR-BRE1a.TIC7382_3.nno_Mc:1, encoding a CR-BRE1a.TIC7382_3.nno_Mc:1protein having an N-terminal and C-terminal truncation relative to theTIC7382 protein, wherein an additional alanine codon is insertedimmediately following the initiating methionine codon, and which is usedfor expression in plant cells.

SEQ ID NO:83 is the amino acid sequence of theCR-BRE1a.TIC7382_3.nno_Mc:1 protein, wherein an additional alanine aminoacid is inserted immediately following the initiating methionine, andcomprises amino acids 52 through 659 of TIC7382.

SEQ ID NO:84 is a synthetic coding sequence,CR-BRE1a.TIC7383_19.nno_Mc:1, encoding a CR-BRE1a.TIC7383_19.nno_Mc:1protein having an N-terminal and C-terminal truncation relative to theTIC7383 protein, and which is used for expression in plant cells.

SEQ ID NO:85 is the amino acid sequence of theCR-BRE1a.TIC7383_19.nno_Mc:1 protein, and comprises amino acids 15through 668 of TIC7383.

SEQ ID NO:86 is a synthetic coding sequence,CR-BRE1a.TIC7383_20.nno_Mc:1, encoding a CR-BRE1a.TIC7383_20.nno_Mc:1protein having an N-terminal and C-terminal truncation relative to theTIC7383 protein, and which is used for expression in plant cells.

SEQ ID NO:87 is the amino acid sequence of theCR-BRE1a.TIC7383_20.nno_Mc:1 protein, and comprises amino acids 15through 661 of TIC7383.

SEQ ID NO:88 is a synthetic coding sequence,CR-BRE1a.TIC7383_21.nno_Mc:1, encoding a CR-BRE1a.TIC7383_21.nno_Mc:1protein having an N-terminal and C-terminal truncation relative to theTIC7383 protein, wherein an additional alanine codon is insertedimmediately following the initiating methionine codon, and which is usedfor expression in plant cells.

SEQ ID NO:89 is the amino acid sequence of theCR-BRE1a.TIC7383_21.nno_Mc:1 protein, wherein an additional alanineamino acid is inserted immediately following the initiating methionine,and comprises amino acids 54 through 661 of TIC7383.

SEQ ID NO:90 is a synthetic coding sequence,CR-BRE1a.TIC7383_22.nno_Mc:1, encoding a CR-BRE1a.TIC7383_22.nno_Mc:1protein having an N-terminal and C-terminal truncation relative to theTIC7383 protein, and which is used for expression in plant cells.

SEQ ID NO:91 is the amino acid sequence of theCR-BRE1a.TIC7383_22.nno_Mc:1 protein, and comprises amino acids 54through 668 of TIC7383.

SEQ ID NO:92 is a synthetic coding sequence,CR-BRE1a.TIC7383_23.nno_Mc:1, encoding a CR-BRE1a.TIC7383_23.nno_Mc:1protein having an N-terminal and C-terminal truncation relative to theTIC7383 protein, and which is used for expression in plant cells.

SEQ ID NO:93 is the amino acid sequence of theCR-BRE1a.TIC7383_23.nno_Mc:1 protein, and comprises amino acids 54through 661 of TIC7383.

SEQ ID NO:94 is a synthetic coding sequence,CR-BRE1a.TIC7383_24.nno_Mc:2, encoding a CR-BRE1a.TIC7383_24.nno_Mc:2protein having an N-terminal and C-terminal truncation relative to theTIC7383 protein, and which is used for expression in plant cells.

SEQ ID NO:95 is the amino acid sequence of theCR-BRE1a.TIC7383_24.nno_Mc:2 protein, and comprises amino acids 73through 661 of TIC7383.

SEQ ID NO:96 is a synthetic coding sequence,CR-BRE1a.TIC7383_25.nno_Mc:3, encoding a CR-BRE1a.TIC7383_25.nno_Mc:3protein having an N-terminal and C-terminal truncation relative to theTIC7383 protein, and which is used for expression in plant cells.

SEQ ID NO:97 is the amino acid sequence of theCR-BRE1a.TIC7383_25.nno_Mc:3 protein, and comprises amino acids 94through 661 of TIC7383.

SEQ ID NO:98 is a synthetic coding sequence,CR-BRE1a.TIC7383_26.nno_Mc:1, encoding a CR-BRE1a.TIC7383_26.nno_Mc:1protein having an N-terminal and C-terminal truncation relative to theTIC7383 protein, and which is used for expression in plant cells.

SEQ ID NO:99 is the amino acid sequence of theCR-BRE1a.TIC7383_26.nno_Mc:1 protein, and comprises amino acids 114through 661 of TIC7383.

SEQ ID NO:100 is a synthetic coding sequence,CR-BRE1a.TIC7383_27.nno_Mc:1, encoding a CR-BRE1a.TIC7383_27.nno_Mc:1protein having an N-terminal and C-terminal truncation relative to theTIC7383 protein, wherein an additional alanine codon is insertedimmediately following the initiating methionine codon, and which is usedfor expression in plant cells.

SEQ ID NO:101 is the amino acid sequence of theCR-BRE1a.TIC7383_27.nno_Mc:1 protein, wherein an additional alanineamino acid is inserted immediately following the initiating methionine,and comprises amino acids 54 through 658 of TIC7383.

SEQ ID NO:102 is a synthetic coding sequence,CR-BRE1a.TIC7383_28.nno_Mc:1, encoding a CR-BRE1a.TIC7383_28.nno_Mc:1protein having an N-terminal and C-terminal truncation relative to theTIC7383 protein, and which is used for expression in plant cells.

SEQ ID NO:103 is the amino acid sequence of theCR-BRE1a.TIC7383_28.nno_Mc:1 protein, and comprises amino acids 15through 658 of TIC7383.

SEQ ID NO:104 is a synthetic coding sequence,CR-BRE1a.TIC7383_29.nno_Mc:1, encoding a CR-BRE1a.TIC7383_29.nno_Mc:1protein having a C-terminal truncation relative to the TIC7383 protein,wherein an additional alanine codon is inserted immediately followingthe initiating methionine codon, and which is used for expression inplant cells.

SEQ ID NO:105 is the amino acid sequence of theCR-BRE1a.TIC7383_29.nno_Mc:1 protein, wherein an additional alanineamino acid is inserted immediately following the initiating methionine,and comprises amino acids 1 through 963 of TIC7383.

SEQ ID NO:106 is a synthetic coding sequence,CR-BRE1a.TIC7383_30.nno_Mc:1, encoding a CR-BRE1a.TIC7383_30.nno_Mc:1protein, wherein an additional alanine codon is inserted immediatelyfollowing the initiating methionine codon, and comprising mutations tothe codons corresponding amino acid positions 964, 966, and 968 relativeto TIC7383, and which is used for expression in plant cells.

SEQ ID NO:107 is the amino acid sequence of theCR-BRE1a.TIC7383_30.nno_Mc:1 protein, wherein an additional alanineamino acid is inserted immediately following the initiating methionine,and comprises the mutations, K964A; R966A; K968A, relative to TIC7383.

SEQ ID NO:108 is a synthetic coding sequence,CR-BRE1a.TIC7383_31.nno_Mc:1, encoding a CR-BRE1a.TIC7383_31.nno_Mc:1protein having a C-terminal truncation relative to the TIC7383 protein,wherein an additional alanine codon is inserted immediately followingthe initiating methionine codon, and comprising mutations to the codonscorresponding amino acid positions 964, 966, and 968 relative toTIC7383, and which is used for expression in plant cells.

SEQ ID NO:109 is the amino acid sequence of theCR-BRE1a.TIC7383_31.nno_Mc:1 protein, wherein an additional alanineamino acid is inserted immediately following the initiating methionine,and comprising amino acids 1 through 1065, and also comprises themutations, K964A; R966A; K968A, relative to TIC7383.

SEQ ID NO:110 is a synthetic coding sequence,CR-BRE1a.TIC7383_32.nno_Mc:1, encoding a CR-BRE1a.TIC7383_32.nno_Mc:1protein, wherein an additional alanine codon is inserted immediatelyfollowing the initiating methionine codon, and comprising a deletion ofthe codons corresponding amino acid positions 964 through 969 relativeto TIC7383, and which is used for expression in plant cells.

SEQ ID NO:111 is the amino acid sequence of theCR-BRE1a.TIC7383_32.nno_Mc:1 protein, wherein an additional alanineamino acid is inserted immediately following the initiating methionine,and comprising a deletion of amino acids 964 through 969 relative toTIC7383.

SEQ ID NO:112 is a synthetic coding sequence, GOI-TIC10743.nno_Mc:1,encoding a GOI-TIC10743.nno_Mc:1 chimeric protein comprised of domainsone and two of TIC7383 and domain three of TIC7042.

SEQ ID NO:113 is the amino acid sequence of the GOI-TIC10743.nno_Mc:1chimeric protein.

SEQ ID NO:114 is a synthetic coding sequence, GOI-TIC10744.nno_Mc:1,encoding a GOI-TIC10744.nno_Mc:1 chimeric protein comprised of domainsone and two of TIC7383 and domain three of TIC7381.

SEQ ID NO:115 is the amino acid sequence of the GOI-TIC10744.nno_Mc:1chimeric protein.

SEQ ID NO:116 is a synthetic coding sequence, GOI-TIC10745.nno_Mc:1,encoding a GOI-TIC10745.nno_Mc:1 chimeric protein comprised of domainsone and two of TIC7383 and domain three of TIC7382.

SEQ ID NO:117 is the amino acid sequence of the GOI-TIC10745.nno_Mc:1chimeric protein.

SEQ ID NO:118 is a synthetic coding sequence, GOI-TIC10746.nno_Mc:1,encoding a GOI-TIC10746.nno_Mc:1 chimeric protein comprised of domainsone and two of TIC7382 and domain three of TIC7383.

SEQ ID NO:119 is the amino acid sequence of the GOI-TIC10746.nno_Mc:1chimeric protein.

SEQ ID NO:120 is a synthetic coding sequence, GOI-TIC10747.nno_Mc:1,encoding a GOI-TIC10747.nno_Mc:1 chimeric protein comprised of domainsone and two of TIC7381 and domain three of TIC7383.

SEQ ID NO:121 is the amino acid sequence of the GOI-TIC10747.nno_Mc:1chimeric protein.

SEQ ID NO:122 is a synthetic coding sequence, GOI-TIC10748.nno_Mc:1,encoding a GOI-TIC10748.nno_Mc:1 chimeric protein comprised of domainsone and two of TIC7042 and domain three of TIC7383.

SEQ ID NO:123 is the amino acid sequence of the GOI-TIC10748.nno_Mc:1chimeric protein.

SEQ ID NO:124 is a synthetic coding sequence, TIC10746NTermExt1,encoding a TIC10746NTermExt1 chimeric protein comprised of domains oneand two of TIC7382 and domain three of TIC7383 which also includes theN-terminal extension peptide derived from TIC7382. The N-terminalextension of TIC7382 comprises amino acids 1-51 of the TIC7382 toxinprotein and is encoded by the first 153 nucleotides of the TIC7382coding sequence.

SEQ ID NO:125 is the amino acid sequence of the TIC10746NTermExt1chimeric protein.

SEQ ID NO:126 is a synthetic coding sequence, TIC10746NTermExt2,encoding a TIC10746NTermExt2 chimeric protein comprised of domains oneand two of TIC7382 and domain three of TIC7383 which also includes theN-terminal extension peptide derived from TIC7383. The N-terminalextension of TIC7383 comprises amino acids 1-53 of the TIC7383 toxinprotein and is encoded by the first 159 nucleotides of the TIC7383coding sequence.

SEQ ID NO:127 is the amino acid sequence of the TIC10746NTermExt2chimeric protein.

DETAILED DESCRIPTION OF THE INVENTION

The problem in the art of agricultural pest control can be characterizedas a need for new toxin proteins that are efficacious against targetpests, exhibit broad spectrum toxicity against target pest species, arecapable of being expressed in plants without causing undesirableagronomic issues, and provide an alternative mode of action compared tocurrent toxins that are used commercially in plants.

Novel insecticidal protein classes, exemplified by TIC7040, TIC7040HT,TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, and TIC7389 andrelated family members that provide resistance against Coleopteran andLepidopteran insect pests, and more particularly against corn rootwormpest species, are disclosed. Also disclosed are synthetic codingsequences designed for expression in a plant cell that encode TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, and TIC7383 related familymembers, along with N-terminal and C-terminal truncation variants andchimeras of proteins in TIC7040 toxin class. Further disclosed arerecombinant nucleic acid molecules comprising a promoter in operablelinkage to a coding sequence encoding a TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, or TIC7389 toxin protein,or related family members, or fragments thereof.

Reference in this application to TIC7040, “TIC7040 protein”, “TIC7040protein toxin”, “TIC7040 toxin protein”, “TIC7040 pesticidal protein”,“TIC7040-related toxins”, or “TIC7040-related toxin protein”, and thelike, refer to any novel pesticidal protein or insect inhibitoryprotein, that comprises, that consists of, that is substantiallyhomologous to, that is similar to, or that is derived from anypesticidal protein or insect inhibitory protein sequence of TIC7040 (SEQID NO:2) and pesticidal or insect inhibitory segments thereof, orcombinations thereof, that confer activity against Coleopteran pests orLepidopteran pests, including any protein exhibiting pesticidal orinsect inhibitory activity if alignment of such protein with TIC7040results in amino acid sequence identity of any fraction percentage fromabout 90 to about 100 percent.

Reference in this application to TIC7040HT, “TIC7040HT protein”,“TIC7040HT protein toxin”, “TIC7040HT toxin protein”, “TIC7040HTpesticidal protein”, “TIC7040HT-related toxins”, or “TIC7040HT-relatedtoxin protein”, and the like, refer to any novel pesticidal protein orinsect inhibitory protein, that comprises, that consists of, that issubstantially homologous to, that is similar to, or that is derived fromany pesticidal protein or insect inhibitory protein sequence ofTIC7040HT (SEQ ID NO:4) and pesticidal or insect inhibitory segmentsthereof, or combinations thereof, that confer activity againstColeopteran pests or Lepidopteran pests, including any proteinexhibiting pesticidal or insect inhibitory activity if alignment of suchprotein with TIC7040HT results in amino acid sequence identity of anyfraction percentage from about 90 to about 100 percent.

Reference in this application to TIC7042, “TIC7042 protein”, “TIC7042protein toxin”, “TIC7042 toxin protein”, “TIC7042 pesticidal protein”,“TIC7042-related toxins”, or “TIC7042-related toxin protein”, and thelike, refer to any novel pesticidal protein or insect inhibitoryprotein, that comprises, that consists of, that is substantiallyhomologous to, that is similar to, or that is derived from anypesticidal protein or insect inhibitory protein sequence of TIC7042 (SEQID NO:12) and pesticidal or insect inhibitory segments thereof, orcombinations thereof, that confer activity against Coleopteran pests orLepidopteran pests, including any protein exhibiting pesticidal orinsect inhibitory activity if alignment of such protein with TIC7042results in amino acid sequence identity of any fraction percentage fromabout 93 to about 100 percent.

Reference in this application to TIC7381, “TIC7381 protein”, “TIC7381protein toxin”, “TIC7381 toxin protein”, “TIC7381 pesticidal protein”,“TIC7381-related toxins”, or “TIC7381-related toxin protein”, and thelike, refer to any novel pesticidal protein or insect inhibitoryprotein, that comprises, that consists of, that is substantiallyhomologous to, that is similar to, or that is derived from anypesticidal protein or insect inhibitory protein sequence of TIC7381 (SEQID NO:14) and pesticidal or insect inhibitory segments thereof, orcombinations thereof, that confer activity against Coleopteran pests orLepidopteran pests, including any protein exhibiting pesticidal orinsect inhibitory activity if alignment of such protein with TIC7381results in amino acid sequence identity of any fraction percentage fromabout 90 to about 100 percent.

Reference in this application to TIC7382, “TIC7382 protein”, “TIC7382protein toxin”, “TIC7382 toxin protein”, “TIC7382 pesticidal protein”,“TIC7382-related toxins”, or “TIC7382-related toxin protein”, and thelike, refer to any novel pesticidal protein or insect inhibitoryprotein, that comprises, that consists of, that is substantiallyhomologous to, that is similar to, or that is derived from anypesticidal protein or insect inhibitory protein sequence of TIC7382 (SEQID NO:16) and pesticidal or insect inhibitory segments thereof, orcombinations thereof, that confer activity against Coleopteran pests orLepidopteran pests, including any protein exhibiting pesticidal orinsect inhibitory activity if alignment of such protein with TIC7382results in amino acid sequence identity of any fraction percentage fromabout 93 to about 100 percent.

Reference in this application to TIC7383, “TIC7383 protein”, “TIC7383protein toxin”, “TIC7383 toxin protein”, “TIC7383 pesticidal protein”,“TIC7383-related toxins”, or “TIC7383-related toxin protein”, and thelike, refer to any novel pesticidal protein or insect inhibitoryprotein, that comprises, that consists of, that is substantiallyhomologous to, that is similar to, or that is derived from anypesticidal protein or insect inhibitory protein sequence of TIC7383 (SEQID NO:18) and pesticidal or insect inhibitory segments thereof, orcombinations thereof, that confer activity against Coleopteran pests orLepidopteran pests, including any protein exhibiting pesticidal orinsect inhibitory activity if alignment of such protein with TIC7383results in amino acid sequence identity of any fraction percentage fromabout 75 to about 100 percent.

Reference in this application to TIC7386, “TIC7386 protein”, “TIC7386protein toxin”, “TIC7386 toxin protein”, “TIC7386 pesticidal protein”,“TIC7386-related toxins”, or “TIC7386-related toxin protein”, and thelike, refer to any novel pesticidal protein or insect inhibitoryprotein, that comprises, that consists of, that is substantiallyhomologous to, that is similar to, or that is derived from anypesticidal protein or insect inhibitory protein sequence of TIC7386 (SEQID NO:30) and pesticidal or insect inhibitory segments thereof, orcombinations thereof, that confer activity against Coleopteran pests orLepidopteran pests, including any protein exhibiting pesticidal orinsect inhibitory activity if alignment of such protein with TIC7386results in amino acid sequence identity of any fraction percentage fromabout 75 to about 100 percent.

Reference in this application to TIC7388, “TIC7388 protein”, “TIC7388protein toxin”, “TIC7388 toxin protein”, “TIC7388 pesticidal protein”,“TIC7388-related toxins”, or “TIC7388-related toxin protein”, and thelike, refer to any novel pesticidal protein or insect inhibitoryprotein, that comprises, that consists of, that is substantiallyhomologous to, that is similar to, or that is derived from anypesticidal protein or insect inhibitory protein sequence of TIC7388 (SEQID NO:32) and pesticidal or insect inhibitory segments thereof, orcombinations thereof, that confer activity against Coleopteran pests orLepidopteran pests, including any protein exhibiting pesticidal orinsect inhibitory activity if alignment of such protein with TIC7388results in amino acid sequence identity of any fraction percentage fromabout 99 to about 100 percent.

Reference in this application to TIC7389, “TIC7389 protein”, “TIC7389protein toxin”, “TIC7389 toxin protein”, “TIC7389 pesticidal protein”,“TIC7389-related toxins”, or “TIC7389-related toxin protein”, and thelike, refer to any novel pesticidal protein or insect inhibitoryprotein, that comprises, that consists of, that is substantiallyhomologous to, that is similar to, or that is derived from anypesticidal protein or insect inhibitory protein sequence of TIC7389 (SEQID NO:34) and pesticidal or insect inhibitory segments thereof, orcombinations thereof, that confer activity against Coleopteran pests orLepidopteran pests, including any protein exhibiting pesticidal orinsect inhibitory activity if alignment of such protein with TIC7389results in amino acid sequence identity of any fraction percentage fromabout 90 to about 100 percent.

The term “segment” or “fragment” is used in this application to describeconsecutive amino acid or nucleic acid sequences that are shorter thanthe complete amino acid or nucleic acid sequence describing a TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, orTIC7389 protein or related family member insecticidal protein. Inspecific embodiments, fragments of a TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, or TIC7389 protein areprovided comprising at least about 50, at least about 75, at least about95, at least about 100, at least about 125, at least about 150, at leastabout 175, at least about 200, at least about 225, at least about 250,at least about 275, at least about 300, at least about 500, at leastabout 600, at least about 700, at least about 750, at least about 800,at least about 850, at least about 900, at least about 950, at leastabout 1000, at least about 1050, at least about 1100, at least about1150, at least about 1200, at least about 1250, at least about 1300, orat least about 1350 contiguous amino acids, or longer, of a proteinhaving insecticidal activity as disclosed herein. In certainembodiments, the invention provides fragments of a protein providedherein, having the activity of the full length sequence. Methods forproducing such fragments from a starting molecule are well known in theart.

A segment or fragment exhibiting insect inhibitory activity is alsodisclosed in this application if alignment of such segment or fragment,with the corresponding section of the TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, or TIC7389 protein setforth in SEQ ID NOs:2, 4, 12, 14, 16, 18, 20, 30, 32, and 34, results inamino acid sequence identity of any fraction percentage from about 75 toabout 100 percent between the segment or fragment and the correspondingsection of the TIC7383 and TIC7386 proteins; or respectively results inamino acid sequence identity of any fraction percentage from about 90 toabout 100 percent between the segment or fragment and the correspondingsection of the TIC7040, TIC7040HT, TIC7381, and TIC7389 proteins; orrespectively results in amino acid sequence identity of any fractionpercentage from about 93 to about 100 percent between the segment orfragment and the corresponding section of the TIC7042 and TIC7382proteins; or respectively results in amino acid sequence identity of anyfraction percentage from about 99 to about 100 percent between thesegment or fragment and the corresponding section of the TIC7388protein.

Reference in this application to the terms “active” or “activity”,“pesticidal activity” or “pesticidal” or “insecticidal activity”,“insect inhibitory” or “insecticidal” refer to efficacy of a toxicagent, such as a protein toxin, in inhibiting (inhibiting growth,feeding, fecundity, or viability), suppressing (suppressing growth,feeding, fecundity, or viability), controlling (controlling the pestinfestation, controlling the pest feeding activities on a particularcrop containing an effective amount of the TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, or TIC7389 protein orrelated family member insecticidal protein) or killing (causing themorbidity, mortality, or reduced fecundity of) a pest. These terms areintended to include the result of providing a pesticidally effectiveamount of a toxic protein to a pest where the exposure of the pest tothe toxic protein results in morbidity, mortality, reduced fecundity, orstunting. These terms also include repulsion of the pest from the plant,a tissue of the plant, a plant part, seed, plant cells, or from theparticular geographic location where the plant may be growing, as aresult of providing a pesticidally effective amount of the toxic proteinin or on the plant. In general, pesticidal activity refers to theability of a toxic protein to be effective in inhibiting the growth,development, viability, feeding behavior, mating behavior, fecundity, orany measurable decrease in the adverse effects caused by an insectfeeding on this protein, protein fragment, protein segment orpolynucleotide of a particular target pest, including but not limited toinsects of the order Lepidoptera, Coleoptera or Hemiptera. The toxicprotein can be produced by the plant or can be applied to the plant orto the environment within the location where the plant is located. Theterms “bioactivity”, “effective”, “efficacious” or variations thereofare also terms interchangeably utilized in this application to describethe effects of proteins of the present invention on target insect pests.

A pesticidally effective amount of a toxic agent, when provided in thediet of a target pest, exhibits pesticidal activity when the toxic agentcontacts the pest. A toxic agent can be a pesticidal protein or one ormore chemical agents known in the art. Pesticidal or insecticidalchemical agents and pesticidal or insecticidal protein agents can beused alone or in combinations with each other. Chemical agents include,but are not limited to, dsRNA molecules targeting specific genes forsuppression in a target pest, organochlorides, organophosphates,carbamates, pyrethroids, neonicotinoids, and ryanoids. Pesticidal orinsecticidal protein agents include the protein toxins set forth in thisapplication, as well as other proteinaceous toxic agents including thosethat target Lepidopteran and Coleopteran pest species, as well asprotein toxins that are used to control other plant pests such as Cryproteins available in the art for use in controlling Homopteran orHemipteran species.

It is intended that reference to a pest, particularly a pest of a cropplant, means insect pests of crop plants, particularly those that arecontrolled by a TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383,TIC7386, TIC7388, or TIC7389 protein or related family memberinsecticidal protein. Reference to a pest can also include Homopteranand Hemipteran insect pests of plants, as well as nematodes and fungiwhen toxic agents targeting these pests are co-localized or presenttogether with a TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383,TIC7388, TIC7389 proteins or related family member insecticidal protein,or a protein that is about 75 to about 100 percent identical to TIC7383and TIC7386; or a protein that is about 90 to about 100 percentidentical to TIC7040, TIC7040HT, TIC7381, and TIC7389; or a protein thatis about 93 to about 100 percent identical to TIC7042, TIC7386, andTIC7382; or a protein that is about 99 to about 100 percent identical toTIC7388.

The TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386,TIC7388, TIC7389 and related family member insecticidal proteins exhibitinsecticidal activity towards insect pests from the Coleopteran andLepidopteran insect species, including adults, pupae, larvae, andneonates.

The insects of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers, and heliothines in the Family Noctuidae,e.g., fall armyworm (Spodoptera frugiperda), Beet armyworm (Spodopteraexigua), Black armyworm (Spodoptera exempta), Bertha armyworm (Mamestraconfigurata), Southern armyworm (Spodoptera eridania), Black cutworm(Agrotis ipsilon), Cabbage looper (Trichoplusia ni), Soybean looper(Pseudoplusia includens), Velvetbean caterpillar (Anticarsiagemmatalis), Green cloverworm (Hypena scabra), Tobacco budworm(Heliothis virescens), Granulate cutworm (Agrotis subterranea), Armyworm(Pseudaletia unipuncta), Western cutworm (Agrotis orthogonia); borers,casebearers, webworms, coneworms, cabbageworms and skeletonizers fromthe Family Pyralidae, e.g., European corn borer (Ostrinia nubilalis),Navel orangeworm (Amyelois transitella), Corn root webworm (Crambuscaliginosellus), Sod webworm (Herpetogramma licarsisalis), Sunflowermoth (Homoeosoma electellum), Lesser cornstalk borer (Elasmopalpuslignosellus); leafrollers, budworms, seed worms, and fruit worms in theFamily Tortricidae, e.g., Codling moth (Cydia pomonella), Grape berrymoth (Endopiza viteana), Oriental fruit moth (Grapholita molesta),Sunflower bud moth (Suleima helianthana); and many other economicallyimportant Lepidoptera, e.g., Diamondback moth (Plutella xylostella),Pink bollworm (Pectinophora gossypiella) and Gypsy moth (Lymantriadispar). Other insect pests of order Lepidoptera include, e.g., Cottonleaf worm (Alabama argillacea), Fruit tree leaf roller (Archipsargyrospila), European leafroller (Archips rosana) and other Archipsspecies, (Chilo suppressalis, Asiatic rice borer, or rice stem borer),Rice leaf roller (Cnaphalocrocis medinalis), Corn root webworm (Crambuscaliginosellus), Bluegrass webworm (Crambus teterrellus), Southwesterncorn borer (Diatraea grandiosella), Surgarcane borer (Diatraeasaccharalis), Spiny bollworm (Earias insulana), Spotted bollworm (Eariasvittella), Old World bollworm (Helicoverpa armigera), Corn earworm(Helicoverpa zea, also known as soybean podworm and cotton bollworm),Western bean cutworm (Striacosta albicosta), European grape vine moth(Lobesia botrana), Citrus leafminer (Phyllocnistis citrella), largewhite butterfly (Pieris brassicae), small white butterfly (Pieris rapae,also known as imported cabbageworm), Tobacco cutworm (Spodoptera litura,also known as cluster caterpillar), and Tomato leafminer (Tutaabsoluta).

The insects of the order Coleoptera include, but are not limited to,Agriotes spp., Anthonomus spp., Atomaria linearis, Chaetocnema tibialis,Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp.,Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrusspp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinusspp., Popillia spp., Psylliodes spp., Rhizopertha spp., Scarabeidae,Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. andTrogoderma spp, particularly when the pest is Western Corn Rootworm(Diabrotica virgifera, WCR), Northern Corn Rootworm (Diabrotica barberi,NCR), Mexican Corn Rootworm (Diabrotica virgifera zeae, MCR), BrazilianCorn Rootworm (Diabrotica balteata, BZR), Southern Corn Rootworm(Diabrotica undecimpunctata howardii, SCR), Colorado potato beetle(Leptinotarsa decemlineata, CPB), a Brazilian Corn Rootworm complex(BCR, consisting of Diabrotica viridula and Diabrotica speciosa),Crucifer Flea Beetle (Phyllotreta cruciferae), Striped Flea Beetle(Phyllotreta striolata), and Western Black Flea Beetle (Phyllotretapusilla).

The insects of the order Hemiptera include, but are not limited to,Stink Bugs of the family Pentatomidae: Green Stink Bugs from the genusChinavia (Chinavia hilaris, Chinavia marginata, and Chinaviapensylvanica), Stink bugs of the genus Chlorochroa (Chlorochroagranulose, Chlorochroa kanei, Chlorochroa ligata, Chlorochroa lineate,Chlorochroa opuntiae, Chlorochroa persimilis, Chlorochroa rossiana,Chlorochroa sayi, Chlorochroa uhleri, Chlorochroa belfragii, Chlorochroafaceta, Chlorochroa osborni, Chlorochroa saucia, and Chlorochroasenilis), Southern Green Stink Bug (Nezara viridula), Stink Bugs fromthe genus Edessa (Edessa meditabunda, Edessa bifida, and Edessaflorida), the Neotropical Brown Stink Bug (Euschistus heros), stink bugsfrom the genus Euschistus (Euschistus acuminatus, Euschistus biformis,Euschistus conspersus, Euschistus crenator, Euschistus egglestoni,Euschistus ictericus, Euschistus inflatus, Euschistus latimarginatus,Euschistus obscures, Euschistus politus, Euschistus quadrator,Euschistus sevus, Euschistus strenuous, Euschistus tristigmus, andEuschistus variolarius), Brown Marmorated Stink Bug (Halyomorpha halys),Red-Shouldered Stink Bug (Thyanta accerra), stink bugs of the genusThyanta (Thyanta calceata, Thyanta custator, Thyanta pallidovirens,Thyanta perditor, Thyanta maculate, and Thyanta pseudocasta), the GreenBelly Stink Bug (Dichelops melacanthus) and other stink bugs of thegenus Dichelops (Dichelops avilapiresi, Dichelops bicolor, Dichelopsdimidatus, Dichelops furcatus, Dichelops furcifrons, Dichelops lobatus,Dichelops miriamae, Dichelops nigrum, Dichelops peruanus, Dichelopsphoenix, and Dichelops saltensis), the Red Banded Stink Bug (Piezodorusguildinni) as well as Piezodorus lituratus; and insects of the family ofPlataspidae such as Kudzu Bug (Megacopta cribraria), Western tarnishedplant bug (Lygus hesperus), and Tarnished plant bug (Lygus lineolaris).

Reference in this application to an “isolated DNA molecule”, or anequivalent term or phrase, is intended to mean that the DNA molecule isone that is present alone or in combination with other compositions, butnot within its natural environment. For example, nucleic acid elementssuch as a coding sequence, intron sequence, untranslated leadersequence, promoter sequence, transcriptional termination sequence, andthe like, that are naturally found within the DNA of the genome of anorganism are not considered to be “isolated” so long as the element iswithin the genome of the organism and at the location within the genomein which it is naturally found. However, each of these elements, andsubparts of these elements, would be “isolated” within the scope of thisdisclosure so long as the element is not within the genome of theorganism and at the location within the genome in which it is naturallyfound. Similarly, a nucleotide sequence encoding an insecticidal proteinor any naturally occurring insecticidal variant of that protein would bean isolated nucleotide sequence so long as the nucleotide sequence wasnot within the DNA of the bacterium from which the sequence encoding theprotein is naturally found. A synthetic nucleotide sequence encoding theamino acid sequence of the naturally occurring insecticidal proteinwould be considered to be isolated for the purposes of this disclosure.For the purposes of this disclosure, any transgenic nucleotide sequence,i.e., the nucleotide sequence of the DNA inserted into the genome of thecells of a plant or bacterium, or present in an extrachromosomal vector,would be considered to be an isolated nucleotide sequence whether it ispresent within the plasmid or similar structure used to transform thecells, within the genome of the plant or bacterium, or present indetectable amounts in tissues, progeny, biological samples or commodityproducts derived from the plant or bacterium.

As described further herein, an open reading frame (ORF) encodingTIC7040 (SEQ ID NO:1), was discovered in DNA obtained from Brevibacilluslaterosporus strain DSC005019, which encodes the protein toxin presentedas SEQ ID NO:2. Upon amplification using primers designed from theTIC7040 ORF, a slightly longer coding sequence was amplified using DNAobtained from Brevibacillus laterosporus strain DSC005019, hereinreferred to as TIC7040HT (SEQ ID NO:3), which encodes the proteinpresented as SEQ ID NO:4. Bioassay using microbial host cell-derivedTIC7040HT protein demonstrated activity against the Coleopteran pestsWestern Corn Rootworm (Diabrotica virgifera virgifera, WCR),Cry3Bb-resistant Western Corn Rootworm (Diabrotica virgifera virgifera,WCRHP), Northern Corn Rootworm (Diabrotica barberi, NCR), Southern CornRootworm (Diabrotica undecimpunctata howardii, SCR), and Colorado potatobeetle (Leptinotarsa decemlineata, CPB), and the Lepidopteran insectpests Corn Earworm (Helicoverpa zea, (CEW) also known as Soybean Podwormand Cotton Bollworm), Diamondback Moth (Plutella xylostella, DBM),European Corn Borer (Ostrinia nubilalis, ECB), Fall Armyworm (Spodopterafrugiperda, FAW), Soybean Looper (Pseudoplusia includes, SBL),Southwestern Corn Borer (Diatraea grandiosella, SWCB), and VelvetbeanCaterpillar (Anticarsia gemmatalis, VBC).

As described further herein, an ORF encoding TIC7042 (SEQ ID NO:11), wasdiscovered in DNA obtained from Brevibacillus laterosporus strainDSC005973 which encodes the protein toxin presented as SEQ ID NO:12.Bioassay using microbial host cell-derived TIC7042 protein demonstratedactivity against the Coleopteran pests WCR, WCRHP, NCR, SCR, and CPB,and the Lepidopteran insect pests BCW, CEW, DBM, FAW, SBL, and VBC.

As described further herein, an ORF encoding TIC7381 (SEQ ID NO:13), wasdiscovered in DNA obtained from Brevibacillus laterosporus strainDSC006713 which encodes the protein toxin presented as SEQ ID NO:14.Bioassay using microbial host cell-derived TIC7381 protein demonstratedactivity against the Coleopteran pests WCR, WCRHP, NCR, SCR, and CPB,and the Lepidopteran insect pests CEW, DBM, ECB, FAW, SBL, SWCB, andVBC.

As described further herein, an ORF encoding TIC7382 (SEQ ID NO:15), wasdiscovered in DNA obtained from Brevibacillus laterosporus strainDSC007657 which encodes the protein toxin presented as SEQ ID NO:16.Bioassay using microbial host cell-derived TIC7382 protein demonstratedactivity against the Coleopteran pests WCR, WCRHP, NCR, SCR, and CPB,and the Lepidopteran insect pests DBM, ECB, SBL, SWCB, and VBC.

As described further herein, an ORF encoding TIC7383 (SEQ ID NO:17), wasdiscovered in DNA obtained from Brevibacillus laterosporus strainDSC008106 which encodes the protein toxin presented as SEQ ID NO:18.Bioassay using microbial host cell-derived TIC7383 protein demonstratedactivity against the Coleopteran pests WCR, WCRHP, NCR, SCR, and CPB,and the Lepidopteran insect pests CEW, DBM, ECB, and VBC.

As described further herein, ORF's encoding TIC7386 (SEQ ID NO:29),TIC7388 (SEQ ID NO:31), and TIC7389 (SEQ ID NO:33) were discovered inDNA obtained from Brevibacillus laterosporus strains DSC007651,DSC007962, and DSC006878, respectively, and encode the proteinspresented herein as SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:34.Bioassay using microbial host cell-derived TIC7389 protein demonstratedactivity against the Lepidopteran insect pest SBL and Coleopteran pestsWCR and CPB.

Further described herein, chymotryptic and tryptic digests of TIC7040HT,and tryptic digests of TIC7383 maintained activity against WCR, relativeto the full length TIC7040HT and TIC7383. In addition, truncations ofTIC7040HT and TIC7383 maintained activity, and in some cases increasedactivity, against WCR and CPB, relative to the full length TIC7040HT andTIC7383.

Further described herein are chimeric toxins comprised of domainsderived from TIC7381, TIC7382, TIC7383, and TIC7042. The chimeric toxin,TIC10743 (SEQ ID NO:113, encoded by SEQ ID NO:112) is comprised ofdomains one and two of TIC7383 and domain three of TIC7042. The chimerictoxin, TIC10744 (SEQ ID NO:115, encoded by SEQ ID NO:114) is comprisedof domains one and two of TIC7383 and domain three of TIC7381. Thechimeric toxin, TIC10745 (SEQ ID NO:117, encoded by SEQ ID NO:116) iscomprised of domains one and two of TIC7383 and domain three of TIC7382.The chimeric toxin, TIC10746 (SEQ ID NO:119, encoded by SEQ ID NO:118)is comprised of domains one and two of TIC7382 and domain three ofTIC7383. The chimeric toxin, TIC10747 (SEQ ID NO:121, encoded by SEQ IDNO:120) is comprised of domains one and two of TIC7381 and domain threeof TIC7383. The chimeric toxin, TIC10748 (SEQ ID NO:123, encoded by SEQID NO:122) is comprised of domains one and two of TIC7042 and domainthree of TIC7383. The chimeric toxin, TIC10746NTermExt1 (SEQ ID NO:125,encoded by SEQ ID NO:124) is comprised of domains one and two ofTIC7382, domain three of TIC7383, and the N-terminal extension ofTIC7382. The N-terminal extension of TIC7382 comprises amino acids 1-51of the TIC7382 toxin protein and is encoded by the first 153 nucleotidesof the TIC7382 coding sequence. The chimeric toxin, TIC10746NTermExt2(SEQ ID NO:127, encoded by SEQ ID NO:126) is comprised of domains oneand two of TIC7382, domain three of TIC7383, and the N-terminalextension of TIC7383. The N-terminal extension of TIC7383 comprisesamino acids 1-53 of the TIC7383 toxin protein and is encoded by thefirst 159 nucleotides of the TIC7383 coding sequence.

For expression in plant cells, TIC7040, TIC7040HT, TIC7042, TIC7381,TIC7382, or TIC7383, or variants, truncation variants, or chimerasthereof can be expressed to reside in the cytosol or targeted to variousorganelles of the plant cell. For example, targeting a protein to thechloroplast may result in increased levels of expressed protein in atransgenic plant while preventing off-phenotypes from occurring.Targeting may also result in an increase in pest resistance efficacy inthe transgenic event. A target peptide or transit peptide is a short(3-70 amino acids long) peptide chain that directs the transport of aprotein to a specific region in the cell, including the nucleus,mitochondria, endoplasmic reticulum (ER), chloroplast, apoplast,peroxisome and plasma membrane. Some target peptides are cleaved fromthe protein by signal peptidases after the proteins are transported. Fortargeting to the chloroplast, proteins contain transit peptides whichare around 40-50 amino acids. For descriptions of the use of chloroplasttransit peptides, see U.S. Pat. Nos. 5,188,642 and 5,728,925. Manychloroplast-localized proteins are expressed from nuclear genes asprecursors and are targeted to the chloroplast by a chloroplast transitpeptide (CTP). Examples of such isolated chloroplast proteins include,but are not limited to, those associated with the small subunit (SSU) ofribulose-1,5,-bisphosphate carboxylase, ferredoxin, ferredoxinoxidoreductase, the light-harvesting complex protein I and protein II,thioredoxin F, enolpyruvyl shikimate phosphate synthase (EPSPS), andtransit peptides described in U.S. Pat. No. 7,193,133. It has beendemonstrated in vivo and in vitro that non-chloroplast proteins may betargeted to the chloroplast by use of protein fusions with aheterologous CTP and that the CTP is sufficient to target a protein tothe chloroplast. Incorporation of a suitable chloroplast transit peptidesuch as the Arabidopsis thaliana EPSPS CTP (CTP2) (See, Klee et al.,Mol. Gen. Genet. 210:437-442, 1987) or the Petunia hybrida EPSPS CTP(CTP4) (See, della-Cioppa et al., Proc. Natl. Acad. Sci. USA83:6873-6877, 1986) has been shown to target heterologous EPSPS proteinsequences to chloroplasts in transgenic plants (See, U.S. Pat. Nos.5,627,061; 5,633,435; and 5,312,910; and EP 0218571; EP 189707; EP508909; and EP 924299). For targeting TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, and TIC7383 and variants, truncation variants andchimeras thereof to the chloroplast, a sequence encoding a chloroplasttransit peptide is placed 5′ in operable linkage and in frame to asynthetic coding sequence encoding the TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, and TIC7383 protein, or variants, truncation variants,or chimeras thereof, that has been designed for optimal expression inplant cells.

It is contemplated that additional toxin protein sequences related toTIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386,TIC7388, and TIC7389 can be created by using the naturally occurringamino acid sequence of TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382,TIC7383, TIC7386, TIC7388, and TIC7389 to create novel proteins and withnovel properties. The TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382,TIC7383, TIC7386, TIC7388, and TIC7389 toxin proteins can be alignedwith other proteins similar to TIC7040, TIC7040HT, TIC7042, TIC7381,TIC7382, TIC7383, TIC7386, TIC7388, and TIC7389 to combine differencesat the amino acid sequence level into novel amino acid sequence variantsand making appropriate changes to the recombinant nucleic acid sequenceencoding the variants.

This disclosure further contemplates that improved variants of theTIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386,TIC7388, and TIC7389 protein toxin classes can be engineered in plantaby using various gene editing methods known in the art. Suchtechnologies used for genome editing include, but are not limited to,ZFN (zinc-finger nuclease), meganucleases, TALEN (Transcriptionactivator-like effector nucleases), and CRISPR (Clustered RegularlyInterspaced Short Palindromic Repeats)/Cas (CRISPR-associated) systems.These genome editing methods can be used to alter the toxin proteincoding sequence transformed within a plant cell to a different toxincoding sequence. Specifically, through these methods, one or more codonswithin the toxin coding sequence is altered to engineer a new proteinamino acid sequence. Alternatively, a fragment within the codingsequence is replaced or deleted, or additional DNA fragments areinserted into the coding sequence, to engineer a new toxin codingsequence. The new coding sequence can encode a toxin protein with newproperties such as increased activity or spectrum against insect pests,as well as provide activity against an insect pest species whereinresistance has developed against the original insect toxin protein. Theplant cell comprising the gene edited toxin coding sequence can be usedby methods known in the art to generate whole plants expressing the newtoxin protein.

It is also contemplated that fragments of the TIC7040, TIC7040HT,TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, and TIC7389proteins or protein variants thereof can be truncated forms wherein oneor more amino acids are deleted from the N-terminal end, C-terminal end,the middle of the protein, or combinations thereof with insectinhibitory activity. These fragments can be naturally occurring orsynthetic variants of TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382,TIC7383, TIC7386, TIC7388, and TIC7389 or protein variants thereof, butshould retain the insect inhibitory activity of TIC7040, TIC7040HT,TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, and TIC7389.

Proteins that resemble the TIC7040, TIC7040HT, TIC7042, TIC7381,TIC7382, TIC7383, TIC7386, TIC7388, and TIC7389 proteins can beidentified by comparison to each other using various computer-basedalgorithms known in the art. For example, amino acid sequence identitiesof proteins related to TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382,TIC7383, TIC7386, TIC7388, and TIC7389 can be analyzed using a Clustal Walignment using these default parameters: Weight matrix: blosum, Gapopening penalty: 10.0, Gap extension penalty: 0.05, Hydrophilic gaps:On, Hydrophilic residues: GPSNDQERK, Residue-specific gap penalties: On(Thompson, et al (1994) Nucleic Acids Research, 22:4673-4680). Percentamino acid identity is further calculated by the product of 100%multiplied by (amino acid identities/length of subject protein). Otheralignment algorithms are also available in the art and provide resultssimilar to those obtained using a Clustal W alignment.

It is intended that a protein exhibiting insect inhibitory activityagainst a Lepidopteran or Coleopteran insect species is related toTIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386,TIC7388, and TIC7389 if alignment of such query protein with TIC7383 orTIC7386 exhibits at least 75% to about 100% amino acid identity alongthe length of the query protein that is about 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity (or anyfraction of a percentage in this range) between query and subjectprotein; or if alignment of such query protein with TIC7040HT, TIC7381,or TIC7389 exhibits at least 90% to about 100% amino acid identity alongthe length of the query protein that is about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity (or anyfraction of a percentage in this range) between query and subjectprotein; or if alignment of such query protein with TIC7042, or TIC7382exhibits at least 93% to about 100% amino acid identity along the lengthof the query protein that is about 93%, 94%, 95%, 96%, 97%, 98%, 99%,100% amino acid sequence identity (or any fraction of a percentage inthis range) between query and subject protein; or if alignment of suchquery protein with TIC7040HT, TIC7381, or TIC7388 exhibits at least 99%to about 100% amino acid identity along the length of the query protein(or any fraction of a percentage in this range) between query andsubject protein.

Exemplary proteins TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382,TIC7383, TIC7386, TIC7388, and TIC7389 were aligned with each otherusing a Clustal W algorithm. A pair-wise matrix of percent amino acidsequence identities for each of the full-length proteins was created, asreported in Table 1. The number of identical amino acids between twosequences is indicated in parenthesis.

TABLE 1 Pair-wise matrix display of exemplary proteins TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, andTIC7389. TIC7042 TIC7386 TIC7388 TIC7382 TIC7040 TIC7040HT TIC7381TIC7389 TIC7383 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID Toxin NO: 12 NO: 30 NO: 32 NO: 16 NO: 2 NO: 4 NO: 14 NO: 34 NO:18 TIC7042 — 99.1 92.3 87.8 85.7 86.3 86.7 86.7 73.3 SEQ ID (1254)(1169) (1111) (1085) (1093) (1097) (1098) (928) NO: 12 TIC7386 98 — 91.586.7 85.1 85.7 86 86.1 72.6 SEQ ID (1254) (1170) (1109) (1089) (1096)(1100) (1101) (928) NO: 30 TIC7388 92.1 92.2 — 93.7 88.8 89.4 89.8 89.976.1 SEQ ID (1169) (1170) (1189) (1127) (1135) (1140) (1141) (966) NO:32 TIC7382 89.2 89 95.4 — 90.6 91.4 91.2 91.5 76.2 SEQ ID (1111) (1109)(1189) (1129) (1139) (1136) (1140) (949) NO: 16 TIC7040 86.2 86.5 89.589.7 — 99.9 98.5 99 76.6 SEQ ID (1085) (1089) (1127) (1129) (1258)(1240) (1247) (965) NO: 2 TIC7040HT 84.4 84.6 87.6 88 97.1 — 96.6 97.274.8 SEQ ID (1093) (1096) (1135) (1139) (1258) (1251) (1259) (969) NO: 4TIC7381 86.4 86.7 89.8 89.5 97.7 98.6 — 99.5 76.5 SEQ ID (1097) (1100)(1140) (1136) (1240) (1251) (1263) (971) NO: 14 TIC7389 83.2 83.5 86.586.4 94.5 95.5 95.8 — 73.9 SEQ ID (1098) (1101) (1141) (1140) (1247)(1259) (1263) (975) NO: 34 TIC7383 73.9 73.9 76.9 75.6 76.8 77.1 77.377.6 — SEQ ID (928) (928) (966) (949) (965) (969) (971) (975) NO: 18

In addition to percent identity, the TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, and TIC7389 proteins canalso be related by primary structure (conserved amino acid motifs), bylength (about 1243 to about 1259 amino acids) and by othercharacteristics. Bioinformatic analysis suggests that TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, andTIC7389 belong to the Cry43 family of proteins. Characteristics of theTIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386,TIC7388, and TIC7389 proteins are reported in Table 2.

TABLE 2 Selected characteristics of the TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, and TIC7389 proteins andrelated family member proteins. No. of No. of Molecular StronglyStrongly No. of No. of Weight Amino Basic (−) Acidic Hydrophobic Polar(in Acid Isoelectric Charge at Amino Amino Amino Amino Protein Daltons)Length Point PH 7.0 Acids Acids Acids Acids TIC7040 142822.03 12595.8063 −13.0 154 151 618 641 TIC7040HT 146821.27 1295 5.8643 −14.0 161156 636 659 TIC7040_4 75400.92 671 7.0053 2.5 68 61 333 338 TIC7040_567268.98 600 7.5498 4.0 61 53 303 297 TIC7040_6 74075.56 660 7.0051 2.567 60 332 338 TIC7040HT_Tryp 65150.72 582 8.1625 5.5 59 50 294 288TIC7040HT_Chymo 66845.58 597 7.8790 4.5 59 51 302 295CR-BREla.TIC7040_1.nno_Mc:1 71805.04 640 6.7230 1.0 62 57 325 315CR-BREla.TIC7040_11.nno_Mc:1 65281.91 583 8.1625 5.5 59 50 295 288CR-BREla.TIC7040_12.nno_Mc:2 74125.45 660 6.5260 0.0 64 60 329 331CR-BREla.TIC7040_13.nno_Mc:1 69983.90 624 7.2157 3.0 62 55 310 314CR-BREla.TIC7040_14.nno_Mc:1 68315.18 611 6.8814 1.5 59 54 311 300TIC7042 143469.54 1266 6.1143 −9.0 158 148 617 649CR-BREla.TIC7042_1.nno_Mc:1 71715.51 637 6.6825 1.0 62 56 312 325CR-BREla.TIC7042_2.nno_Mc:1 73986.03 657 6.9127 2.5 67 59 319 338TIC7381 143793.24 1269 6.2077 −7.0 162 150 626 643CR-BREla.TIC7381_1.nno_Mc:1 143850.30 1270 6.2077 −7.0 162 150 627 643CR-BREla.TIC7381_2.nno_Mc:1 73892.21 659 6.5260 0.0 64 60 331 328CR-BREla.TIC7381_3.nno_Mc:1 68253.10 611 6.8814 1.5 59 54 312 299TIC7382 140890.86 1246 5.7572 −13.0 149 147 614 632CR-BREla.TIC7382_1.nno_Mc:1 140961.94 1247 5.7572 −13.0 149 147 615 632CR-BREla.TIC7382_2.nno_Mc:1 73858.00 660 6.6112 0.5 63 58 329 331CR-BREla.TIC7382_3.nno_Mc:1 67924.61 610 6.9577 2.0 58 52 310 300TIC7383 142470.64 1256 5.5492 −17.5 151 152 627 629 TIC7383_2 140903.051243 5.5471 −17.5 150 151 626 617 TIC7383_3 74526.97 659 5.7950 −3.0 6161 343 316 TIC7383_4 76846.56 679 5.9941 −2.5 66 65 350 329 TIC7383_572959.38 646 5.7903 −3.0 60 60 342 304 TIC7383_6 75278.97 666 5.9913−2.5 65 64 349 317 TIC7383_Tryp 69242.24 614 5.8225 −2.5 57 57 326 288CR-BREla.TIC7383_1.nno_Mc:1 142541.72 1257 5.5492 −17.5 151 152 628 629CR-BREla.TIC7383_7.nno_Mc:1 69545.62 617 5.8225 −2.5 57 57 328 289CR-BREla.TIC7383_8.nno_Mc:1 74772.20 662 5.7950 −3.0 61 61 344 318CR-BREla.TIC7383_9.nno_Mc:1 75669.20 669 5.5878 −4.0 62 63 347 322CR-BREla.TIC7383_19.nno_Mc:1 74030.53 655 5.5815 −4.0 61 62 345 310CR-BREla.TIC7383_20.nno_Mc:1 73133.53 648 5.7903 −3.0 60 60 342 306CR-BREla.TIC7383_21.nno_Mc:1 68648.63 610 6.0997 −1.5 56 55 325 285CR-BREla.TIC7383_22.nno_Mc:1 69474.54 616 5.8225 −2.5 57 57 327 289CR-BREla.TIC7383_23.nno_Mc:1 68577.55 609 6.0997 −1.5 56 55 324 285CR-BREla.TIC7383_24.nno_Mc:2 66643.51 590 6.6807 0.5 55 52 315 275CR-BREla.TIC7383_25.nno_Mc:3 64647.09 569 6.3851 −0.5 54 52 299 270CR-BREla.TIC7383_26.nno_Mc:1 62376.52 549 6.6802 0.5 54 51 287 262CR-BREla.TIC7383_27.nno_Mc:1 68417.42 607 6.0997 −1.5 56 55 324 283CR-BREla.TIC7383_28.nno_Mc:1 72902.33 645 5.7903 −3.0 60 60 341 304CR-BREla.TIC7383_29.nno_Mc:1 108909.43 964 5.0905 −17.0 104 112 489 475CR-BREla.TIC7383_30.nno_Mc:1 142342.42 1257 5.3793 −20.5 148 152 631 626CR-BREla.TIC7383_31.nno_Mc:1 120993.09 1066 5.4956 −13.0 122 124 538 528CR-BREla.TIC7383_32.nno_Mc:1 141715.71 1251 5.3252 −21.5 146 151 628 623TIC7386 144952.03 1279 6.1338 −9.0 161 150 621 658 TIC7388 143562.871269 6.0794 −9.5 157 148 629 640 TIC7389 149398.97 1319 5.6940 −17.5 162160 652 667 GOI-TIC10743.nno_Mc:1 69704.53 617 6.0297 −2.0 57 56 314 303GOI-TIC10744.nno_Mc:1 69405.21 616 6.0308 −2.0 57 56 317 299GOI-TIC10745.nno_Mc:1 69732.59 617 6.2812 −1.0 58 56 314 303GOI-TIC10746.nno_Mc:1 67737.64 610 6.6433 0.5 57 53 324 286GOI-TIC10747.nno_Mc:1 68393.51 612 6.8012 1.0 59 58 323 289GOI-TIC10748.nno_Mc:1 68118.77 609 6.7284 1.0 59 54 312 297TIC10746NTermExt1 73599.95 659 6.3342 −1.0 62 59 342 317TIC10746NTermExt2 73790.14 661 6.3342 −1.0 62 59 342 319

As described further in the Examples of this application, recombinantnucleic acid molecule sequences encoding TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, and TIC7383, and variants, truncation variants, andchimeras thereof were designed for use in plants. Exemplaryplant-optimized recombinant nucleic acid molecule sequences that weredesigned for use in plants are presented in Table 8 of Example 5, alongwith the corresponding nucleotide and protein sequences, description andmodifications.

Expression cassettes and vectors containing these recombinant nucleicacid molecule sequences can be constructed and introduced into corn,soybean, cotton or other plant cells in accordance with transformationmethods and techniques known in the art. For example,Agrobacterium-mediated transformation is described in U.S. PatentApplication Publications 2009/0138985A1 (soybean), 2008/0280361A1(soybean), 2009/0142837A1 (corn), 2008/0282432 (cotton), 2008/0256667(cotton), 2003/0110531 (wheat), 2001/0042257 A1 (sugar beet), U.S. Pat.No. 5,750,871 (canola), U.S. Pat. No. 7,026,528 (wheat), and U.S. Pat.No. 6,365,807 (rice), and in Arencibia et al. (1998) Transgenic Res.7:213-222 (sugarcane), all of which are incorporated herein by referencein their entirety. Transformed cells can be regenerated into transformedplants that express TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, orTIC7383. To test pesticidal activity, bioassays are performed in thepresence of Lepidoptera pest larvae using plant leaf disks obtained fromtransformed plants, as described in the Examples. To test pesticidalactivity against Coleopteran pests, transformed plants of R_(o) and F₁generation are used in root worm assay, as described in the Examples. Totest pesticidal activity against Hemipteran pests, pods, corn ears orleaves of transformed plants are used in assay, either from tissueremoved from the plant or remaining on the plant as described in theExamples.

As an alternative to traditional transformation methods, a DNA sequence,such as a transgene, expression cassette(s), etc., may be inserted orintegrated into a specific site or locus within the genome of a plant orplant cell via site-directed integration. Recombinant DNA construct(s)and molecule(s) of this disclosure may thus include a donor templatesequence comprising at least one transgene, expression cassette, orother DNA sequence for insertion into the genome of the plant or plantcell. Such donor template for site-directed integration may furtherinclude one or two homology arms flanking an insertion sequence (i.e.,the sequence, transgene, cassette, etc., to be inserted into the plantgenome). The recombinant DNA construct(s) of this disclosure may furthercomprise an expression cassette(s) encoding a site-specific nucleaseand/or any associated protein(s) to carry out site-directed integration.These nuclease expressing cassette(s) may be present in the samemolecule or vector as the donor template (in cis) or on a separatemolecule or vector (in trans). Several methods for site-directedintegration are known in the art involving different proteins (orcomplexes of proteins and/or guide RNA) that cut the genomic DNA toproduce a double strand break (DSB) or nick at a desired genomic site orlocus. As understood in the art, during the process of repairing the DSBor nick introduced by the nuclease enzyme, the donor template DNA maybecome integrated into the genome at the site of the DSB or nick. Thepresence of the homology arm(s) in the donor template may promote theadoption and targeting of the insertion sequence into the plant genomeduring the repair process through homologous recombination, although aninsertion event may occur through non-homologous end joining (NHEJ).Examples of site-specific nucleases that may be used include zinc-fingernucleases, engineered or native meganucleases, TALE-endonucleases, andRNA-guided endonucleases (e.g., Cas9 or Cpf1). For methods usingRNA-guided site-specific nucleases (e.g., Cas9 or Cpf1), the recombinantDNA construct(s) will also comprise a sequence encoding one or moreguide RNAs to direct the nuclease to the desired site within the plantgenome.

Recombinant nucleic acid molecule compositions that encode TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, andTIC7389 or related family member insecticidal proteins are contemplated.For example, TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383,TIC7386, TIC7388, and TIC7389 or related family member insecticidalproteins can be expressed with recombinant DNA constructs in which apolynucleotide molecule with an ORF encoding the protein is operablylinked to genetic expression elements such as a promoter and any otherregulatory element necessary for expression in the system for which theconstruct is intended. Non-limiting examples include a plant-functionalpromoter operably linked to the TIC7040, TIC7040HT, TIC7042, TIC7381,TIC7382, TIC7383, TIC7386, TIC7388, and TIC7389 or related family memberinsecticidal protein encoding sequences for expression of the protein inplants or a Bt-functional promoter operably linked to a TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, andTIC7389 or related family member insecticidal protein encoding sequencesfor expression of the protein in a Bt bacterium or other Bacillusspecies. Other elements can be operably linked to the TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, andTIC7389 or related family member insecticidal protein encoding sequencesincluding, but not limited to, enhancers, introns, untranslated leaders,encoded protein immobilization tags (HIS-tag), translocation peptides(i.e., plastid transit peptides, signal peptides), polypeptide sequencesfor post-translational modifying enzymes, ribosomal binding sites, andRNAi target sites. Exemplary recombinant polynucleotide moleculesprovided herewith include, but are not limited to, a heterologouspromoter operably linked to a polynucleotide such as SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45,SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54,SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64,SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74,SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84,SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94,SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104,SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ IDNO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQID NO:124, and SEQ ID NO:126 that encodes a polypeptide or proteinhaving the amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:43, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, and SEQ ID NO:127. A heterologous promoter can also beoperably linked to synthetic DNA coding sequences encoding a plastidtargeted or untargeted TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382,TIC7383, variants, truncation variants, and chimeras thereof anduntargeted TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383,variants, truncation variants, and chimeras thereof or related familymember insecticidal proteins. The codons of a recombinant nucleic acidmolecule encoding for proteins disclosed herein can be substituted bysynonymous codons (known in the art as a silent substitution).

A recombinant DNA construct comprising an encoding sequence for TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, TIC7389or related family member insecticidal protein can further comprise aregion of DNA that encodes for one or more insect inhibitory agentswhich can be configured to concomitantly express or co-express with aDNA sequence encoding a TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382,TIC7383, TIC7386, TIC7388, TIC7389 or related family member insecticidalprotein, a protein different from a TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, TIC7389 or a related familymember insecticidal protein, an insect inhibitory dsRNA molecule, or anancillary protein. Ancillary proteins include, but are not limited to,co-factors, enzymes, binding-partners, or other agents that function toaid in the effectiveness of an insect inhibitory agent, for example, byaiding its expression, influencing its stability in plants, optimizingfree energy for oligomerization, augmenting its toxicity, and increasingits spectrum of activity. An ancillary protein may facilitate the uptakeof one or more insect inhibitory agents, for example, or potentiate thetoxic effects of the toxic agent.

A recombinant DNA construct can be assembled so that all proteins ordsRNA molecules are expressed from one promoter or each protein or dsRNAmolecules is under separate promoter control or some combinationthereof. The TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383,TIC7386, TIC7388, TIC7389 and related family member proteins of thisinvention can be expressed from a multi-gene expression system in whichone or more TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383,TIC7386, TIC7388, TIC7389 or related family member proteins is expressedfrom a common nucleotide segment which also contains other open readingframes and promoters, depending on the type of expression systemselected. For example, a bacterial multi-gene expression system canutilize a single promoter to drive expression of multiply-linked/tandemopen reading frames from within a single operon (i.e., polycistronicexpression). In another example, a plant multi-gene expression systemcan utilize multiply-unlinked expression cassettes each expressing adifferent protein or other agent such as one or more dsRNA molecules.

Recombinant nucleic acid molecules or recombinant DNA constructscomprising a TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383,TIC7386, TIC7388, TIC7389 or related family member protein encodingsequence can be delivered to host cells by vectors, e.g., a plasmid,baculovirus, synthetic chromosome, virion, cosmid, phagemid, phage, orviral vector. Such vectors can be used to achieve stable or transientexpression of a TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383,TIC7386, TIC7388, TIC7389 or related family member protein encodingsequence in a host cell, or subsequent expression of the encodedpolypeptide. An exogenous recombinant polynucleotide or recombinant DNAconstruct that comprises a protein encoding sequence and that isintroduced into a host cell is referred herein as a “transgene.”

Transgenic bacteria, transgenic plant cells, transgenic plants, andtransgenic plant parts that contain a recombinant polynucleotide thatexpresses any one or more of the TIC7040, TIC7040HT, TIC7042, TIC7381,TIC7382, TIC7383, TIC7386, TIC7388, TIC7389 or related family memberprotein encoding sequences are provided herein. The term “bacterialcell” or “bacterium” can include, but is not limited to, anAgrobacterium, a Bacillus, an Escherichia, a Salmonella, a Pseudomonas,or a Rhizobium cell. The term “plant cell” or “plant” can include but isnot limited to a dicotyledonous cell or a monocotyledonous cell.Contemplated plants and plant cells include, but are not limited to,alfalfa, banana, barley, bean, broccoli, cabbage, brassica, carrot,cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus,coconut, coffee, corn, clover, cotton, a cucurbit, cucumber, Douglasfir, eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce,Loblolly pine, millets, melons, nut, oat, olive, onion, ornamental,palm, pasture grass, pea, peanut, pepper, pigeonpea, pine, potato,poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye,safflower, shrub, sorghum, Southern pine, soybean, spinach, squash,strawberry, sugar beet, sugarcane, sunflower, sweet corn, sweet gum,sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass,watermelon, and wheat plant cell or plant. In certain embodiments,transgenic plants and transgenic plant parts regenerated from atransgenic plant cell are provided. In certain embodiments, thetransgenic plants can be obtained from a transgenic seed, by cutting,snapping, grinding or otherwise disassociating the part from the plant.In certain embodiments, the plant part can be a seed, a boll, a leaf, aflower, a stem, a root, or any portion thereof, or a non-regenerableportion of a transgenic plant part. As used in this context, a“non-regenerable” portion of a transgenic plant part is a portion thatcannot be induced to form a whole plant or that cannot be induced toform a whole plant that is capable of sexual and/or asexualreproduction. In certain embodiments, a non-regenerable portion of aplant part is a portion of a transgenic seed, boll, leaf, flower, stem,or root.

Methods of making transgenic plants that comprise insect, Coleoptera- orLepidoptera-inhibitory amounts of a TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, TIC7389, variant,truncation variant, chimera or related family member protein areprovided. Such plants can be made by introducing a recombinantpolynucleotide that encodes any TIC7040, TIC7040HT, TIC7042, TIC7381,TIC7382, TIC7383, TIC7386, TIC7388, TIC7389, variant, truncationvariant, chimera or related family member protein provided in thisapplication into a plant cell, and selecting a plant derived from saidplant cell that expresses an insect, Coleoptera- orLepidoptera-inhibitory amount of the proteins. Plants can be derivedfrom the plant cells by regeneration, seed, pollen, or meristemtransformation techniques. Methods for transforming plants are known inthe art.

Processed plant products, wherein the processed product comprises adetectable amount of a TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382,TIC7383, TIC7386, TIC7388, TIC7389, variant, truncation variant, chimeraor related family member protein, an insect inhibitory segment orfragment thereof, or any distinguishing portion thereof, are alsodisclosed in this application. In certain embodiments, the processedproduct is selected from the group consisting of plant parts, plantbiomass, oil, meal, sugar, animal feed, flour, flakes, bran, lint,hulls, processed seed, and seed. In certain embodiments, the processedproduct is non-regenerable. The plant product can comprise commodity orother products of commerce derived from a transgenic plant or transgenicplant part, where the commodity or other products can be tracked throughcommerce by detecting nucleotide segments or expressed RNA or proteinsthat encode or comprise distinguishing portions of a TIC7040, TIC7040HT,TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, TIC7389, variant,truncation variant, chimera or related family member protein.

Plants expressing a TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382,TIC7383, TIC7386, TIC7388, TIC7389, variant, truncation variant, chimeraor related family member protein can be crossed by breeding withtransgenic events expressing other toxin proteins and/or expressingother transgenic traits such as herbicide tolerance genes, genesconferring yield or stress tolerance traits, and the like, or suchtraits can be combined in a single vector so that the traits are alllinked.

As described further in the Examples, sequences encoding TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, and TIC7383, variants ofTIC7040HT, TIC7042, TIC7381, TIC7382, and TIC7383, truncation variantsof TIC7040HT, TIC7042, TIC7381, TIC7382, and TIC7383, and chimeras ofTIC7383, TIC7042, TIC7381 and TIC7382 were designed for use in plantsand are presented in Table 8 in Example 5.

Expression cassettes and vectors containing these synthetic orartificial nucleotide sequences can be constructed and introduced intocorn, cotton, and soybean plant cells in accordance with transformationmethods and techniques which are known in the art. Transformed cells areregenerated into transformed plants that are observed to be expressingTIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, or TIC7383; or variants,truncation variants or chimeras thereof. To test pesticidal activity,bioassays are performed in the presence of Lepidopteran, Coleopteran andHemipteran pests.

As further described in the Examples, sequences encoding TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, variants, truncationvariants, chimeras or related family member proteins and sequenceshaving a substantial percentage identity to these proteins can beidentified using methods known to those of ordinary skill in the artsuch as polymerase chain reaction (PCR), thermal amplification andhybridization. For example, a TIC7040, TIC7040HT, TIC7042, TIC7381,TIC7382, or TIC7383 protein and variants, truncation variants, chimerasor related family member proteins can be used to produce antibodies thatbind specifically to related proteins, and can be used to screen for andto find other protein members that are closely related.

Furthermore, nucleotide sequences encoding TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, or TIC7383 and variants, truncation variants, chimerasor related family member proteins can be used as probes and primers forscreening to identify other members of the class using thermal-cycle orisothermal amplification and hybridization methods. For example,oligonucleotides derived from sequences as set forth as SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47,SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56,SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66,SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82,SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92,SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102,SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ IDNO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQID NO:122, SEQ ID NO:124, and SEQ ID NO:126 can be used to determine thepresence or absence of a TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382,or TIC7383 protein, or variants, truncation variants, chimeras, orrelated family member protein transgene in a deoxyribonucleic acidsample derived from a commodity product. Given the sensitivity ofcertain nucleic acid detection methods that employ oligonucleotides, itis anticipated that oligonucleotides derived from the sequences as setforth as SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ IDNO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118,SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, and SEQ ID NO:126 can beused to detect a TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, orTIC7383, variants, truncation variants, or chimeric transgenes thereofin commodity products derived from pooled sources where only a fractionof the commodity product is derived from a transgenic plant containingany of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ IDNO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118,SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, or SEQ ID NO:126. It isfurther recognized that such oligonucleotides can be used to introducenucleotide sequence variation in SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ IDNO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ IDNO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ IDNO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,or SEQ ID NO:126. Such “mutagenesis” oligonucleotides are useful foridentification of TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382,TIC7383, TIC7386, TIC7388, TIC7389 or related family member amino acidsequence variants exhibiting a range of insect inhibitory activity orvaried expression in transgenic plant host cells.

Nucleotide sequence homologs, e.g., insecticidal proteins encoded bynucleotide sequences that hybridize to each or any of the sequencesdisclosed in this application under hybridization conditions, are alsoan embodiment of the present invention. The invention also provides amethod for detecting a first nucleotide sequence that hybridizes to asecond nucleotide sequence, wherein the first nucleotide sequence (orits reverse complement sequence) encodes a pesticidal protein orpesticidal fragment thereof and hybridizes under stringent hybridizationconditions to the second nucleotide sequence. In such case, the secondnucleotide sequence can be the nucleotide sequence selected from thegroup consisting of: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ IDNO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118,SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, and SEQ ID NO:126 understringent hybridization conditions. Nucleotide coding sequenceshybridize to one another under appropriate hybridization conditions andthe proteins encoded by these nucleotide sequences cross react withantiserum raised against any one of the other proteins. Stringenthybridization conditions, as defined herein, comprise at leasthybridization at 42° C. followed by two washes for five minutes each atroom temperature with 2×SSC, 0.1% SDS, followed by two washes for thirtyminutes each at 65° C. in 0.5×SSC, 0.1% SDS. Washes at even highertemperatures constitute even more stringent conditions, e.g.,hybridization conditions of 68° C., followed by washing at 68° C., in2×SSC containing 0.1% SDS.

One skilled in the art will recognize that, due to the redundancy of thegenetic code, many other sequences are capable of encoding proteinsrelated to TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383,TIC7386, TIC7388, or TIC7389, and those sequences, to the extent thatthey function to express pesticidal proteins either in Bacillus strainsor in plant cells, are embodiments of the present invention, recognizingof course that many such redundant coding sequences will not hybridizeunder these conditions to the native Brevibacillus sequences encodingTIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386,TIC7388, or TIC7389. This application contemplates the use of these andother identification methods known to those of ordinary skill in theart, to identify TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383,TIC7386, TIC7388, TIC7389 or related family member protein-encodingsequences and sequences having a substantial percentage identitythereto.

Methods of controlling insects, in particular Lepidoptera or Coleopterainfestations of crop plants, with the TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, or TIC7389 or relatedfamily member proteins are also disclosed in this application. Suchmethods can comprise growing a plant comprising an insect-, Coleoptera-,or Lepidoptera-inhibitory amount of a TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, TIC7389, variant,truncation variant, chimera or related family member toxin protein. Incertain embodiments, such methods can further comprise any one or moreof: (i) applying any composition comprising or encoding a TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388,TIC7389, variant, truncation variant, chimera or related family membertoxin protein to a plant or a seed that gives rise to a plant; and (ii)transforming a plant or a plant cell that gives rise to a plant with apolynucleotide encoding a TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382,TIC7383, TIC7386, TIC7388, TIC7389, variant, truncation variant, chimeraor related family member toxin protein. In general, it is contemplatedthat a TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386,TIC7388, TIC7389, variant, truncation variant or related family membertoxin protein can be provided in a composition, provided in amicroorganism, or provided in a transgenic plant to confer insectinhibitory activity against Lepidopteran or Coleopteran insects.

In certain embodiments, a recombinant nucleic acid molecule of aTIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386,TIC7388, TIC7389, variant, truncation variant, chimera or related familymember toxin protein is the insecticidally active ingredient of aninsect inhibitory composition prepared by culturing recombinant Bacillusor any other recombinant bacterial cell transformed to express theprotein. Such a composition can be prepared by desiccation,lyophilization, homogenization, extraction, filtration, centrifugation,sedimentation, or concentration of a culture of such recombinant cellsexpressing/producing said recombinant polypeptide. Such a process canresult in a Bacillus or other entomopathogenic bacterial cell extract,cell suspension, cell homogenate, cell lysate, cell supernatant, cellfiltrate, or cell pellet. By obtaining the recombinant polypeptides soproduced, a composition that includes the recombinant polypeptides caninclude bacterial cells, bacterial spores, and parasporal inclusionbodies and can be formulated for various uses, including as agriculturalinsect inhibitory spray products or as insect inhibitory formulations indiet bioassays.

In one embodiment, to reduce the likelihood of resistance development,an insect inhibitory composition comprising TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, TIC7389, variant,truncation variant, chimera or related family member can furthercomprise at least one additional polypeptide known to those of ordinaryskill in the art that exhibits insect inhibitory activity against thesame Lepidopteran or Coleopteran insect species, but which is differentfrom the TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383,TIC7386, TIC7388, TIC7389, variant, truncation variant, chimera orrelated family member toxin protein. Possible additional polypeptidesfor such a composition include an insect inhibitory protein and aninsect inhibitory dsRNA molecule. One example for the use of suchribonucleotide sequences to control insect pests is described in Baum,et al. (U.S. Patent Publication 2006/0021087 A1).

Such additional polypeptide for the control of Lepidopteran pests may beselected from the group consisting of an insect inhibitory protein, suchas, but not limited to, Cry1A (U.S. Pat. No. 5,880,275), Cry1Ab, Cry1Ac,Cry1A.105, Cry1Ae, Cry1B (U.S. patent publication Ser. No. 10/525,318),Cry1C (U.S. Pat. No. 6,033,874), Cry1D, Cry1Da and variants thereof,Cry1E, Cry1F, and Cry1A/F chimeras (U.S. Pat. Nos. 7,070,982; 6,962,705;and 6,713,063), Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry1-typechimeras such as, but not limited to, TIC836, TIC860, TIC867, TIC869,and TIC1100 (International Application Publication WO2016/061391 (A2)),TIC2160 (International Application Publication WO2016/061392(A2)),Cry2A, Cry2Ab (U.S. Pat. No. 7,064,249), Cry2Ae, Cry4B, Cry6, Cry7,Cry8, Cry9, Cry15, Cry43A, Cry43B, Cry51Aa1, ET66, TIC400, TIC800,TIC834, TIC1415, Vip3A, VIP3Ab, VIP3B, AXMI-001, AXMI-002, AXMI-030,AXMI-035, AND AXMI-045 (U.S. Patent Publication 2013-0117884 A1),AXMI-52, AXMI-58, AXMI-88, AXMI-97, AXMI-102, AXMI-112, AXMI-117,AXMI-100 (U.S. Patent Publication 2013-0310543 A1), AXMI-115, AXMI-113,AXMI-005 (U.S. Patent Publication 2013-0104259 A1), AXMI-134 (U.S.Patent Publication 2013-0167264 A1), AXMI-150 (U.S. Patent Publication2010-0160231 A1), AXMI-184 (U.S. Patent Publication 2010-0004176 A1),AXMI-196, AXMI-204, AXMI-207, AXMI-209 (U.S. Patent Publication2011-0030096 A1), AXMI-218, AXMI-220 (U.S. Patent Publication2014-0245491 A1), AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z, AXMI-225z(U.S. Patent Publication 2014-0196175 A1), AXMI-238 (U.S. PatentPublication 2014-0033363 A1), AXMI-270 (U.S. Patent Publication2014-0223598 A1), AXMI-345 (U.S. Patent Publication 2014-0373195 A1),AXMI-335 (International Application Publication WO2013/134523(A2)),DIG-3 (U.S. Patent Publication 2013-0219570 A1), DIG-5 (U.S. PatentPublication 2010-0317569 A1), DIG-11 (U.S. Patent Publication2010-0319093 A1), AfIP-1A and derivatives thereof (U.S. PatentPublication 2014-0033361 A1), AfIP-1B and derivatives thereof (U.S.Patent Publication 2014-0033361 A1), PIP-1APIP-1B (U.S. PatentPublication 2014-0007292 A1), PSEEN3174 (U.S. Patent Publication2014-0007292 A1), AECFG-592740 (U.S. Patent Publication 2014-0007292A1), Pput_1063 (U.S. Patent Publication 2014-0007292 A1), DIG-657(International Application Publication WO2015/195594(A2)), Pput_1064(U.S. Patent Publication 2014-0007292 A1), GS-135 and derivativesthereof (U.S. Patent Publication 2012-0233726 A1), GS153 and derivativesthereof (U.S. Patent Publication 2012-0192310 A1), GS154 and derivativesthereof (U.S. Patent Publication 2012-0192310 A1), GS155 and derivativesthereof (U.S. Patent Publication 2012-0192310 A1), SEQ ID NO:2 andderivatives thereof as described in U.S. Patent Publication 2012-0167259A1, SEQ ID NO:2 and derivatives thereof as described in U.S. PatentPublication 2012-0047606 A1, SEQ ID NO:2 and derivatives thereof asdescribed in U.S. Patent Publication 2011-0154536 A1, SEQ ID NO:2 andderivatives thereof as described in U.S. Patent Publication 2011-0112013A1, SEQ ID NO:2 and 4 and derivatives thereof as described in U.S.Patent Publication 2010-0192256 A1, SEQ ID NO:2 and derivatives thereofas described in U.S. Patent Publication 2010-0077507 A1, SEQ ID NO:2 andderivatives thereof as described in U.S. Patent Publication 2010-0077508A1, SEQ ID NO:2 and derivatives thereof as described in U.S. PatentPublication 2009-0313721 A1, SEQ ID NO:2 or 4 and derivatives thereof asdescribed in U.S. Patent Publication 2010-0269221 A1, SEQ ID NO:2 andderivatives thereof as described in U.S. Pat. No. 7,772,465 (B2),CF161_0085 and derivatives thereof as described in WO2014/008054 A2,Lepidopteran toxic proteins and their derivatives as described in USPatent Publications US2008-0172762 A1, US2011-0055968 A1, andUS2012-0117690 A1; SEQ ID NO:2 and derivatives thereof as described inU.S. Pat. No. 7,510,878(B2), SEQ ID NO:2 and derivatives thereof asdescribed in U.S. Pat. No. 7,812,129(B1), Cry71Aa1 and Cry72Aa1 (USPatent Publication US2016-0230187 A1), Axmi422 (US Patent PublicationUS2016-0201082 A1), Axmi440 (US Patent Publication US2016-0185830 A1),Axmi281 (US Patent Publication 2016-0177332 A1), BT-0044, BT-0051,BT-0068, BT-0128 and variants thereof (WO 2016-094159 A1), BT-009,BT-0012, BT-0013, BT-0023, BT0067 and variants thereof (WO 2016-094165A1), Cry1JP578V, Cry1JPS1, Cry1 JPS1P578V (WO 2016-061208 A1); and thelike.

Such additional polypeptide for the control of Coleopteran pests may beselected from the group consisting of an insect inhibitory protein, suchas, but not limited to, Cry3Bb (U.S. Pat. No. 6,501,009), Cry1Cvariants, Cry3A variants, Cry3, Cry3B, Cry34/35, 5307, AXMI134 (U.S.Patent Publication 2013-0167264 A1) AXMI-184 (U.S. Patent Publication2010-0004176 A1), AXMI-205 (U.S. Patent Publication 2014-0298538 A1),AXMI-207 (U.S. Patent Publication 2013-0303440 A1), AXMI-218, AXMI-220(U.S. Patent Publication 20140245491A1), AXMI-221z, AXMI-223z (U.S.Patent Publication 2014-0196175 A1), AXMI-279 (U.S. Patent Publication2014-0223599 A1), AXMI-R1 and variants thereof (U.S. Patent Publication2010-0197592 A1, TIC407, TIC417, TIC431, TIC807, TIC853, TIC901,TIC1201, TIC3131, DIG-10 (U.S. Patent Publication 2010-0319092 A1),eHIPs (U.S. Patent Application Publication No. 2010/0017914), 1P3 andvariants thereof (U.S. Patent Publication 2012-0210462 ω-Hexatoxin-Hv1a(U.S. Patent Application Publication 2014-0366227 A1), PHI-4 variants(U.S. Patent Application Publication 2016-0281105 A1), PIP-72 variants(WO 2016-144688 A1), PIP-45 variants, PIP-64 variants, PIP-74 variants,PIP-75 variants, and PIP-77 variants (WO 2016-144686 A1), DIG-305 (WO2016109214 A1), PIP-47 variants (U.S. Patent Publication 2016-0186204A1), DIG-17, DIG-90, DIG-79 (WO 2016-057123 A1), DIG-303 (WO 2016-070079A1); and the like.

In other embodiments, such composition/formulation can further compriseat least one additional polypeptide that exhibits insect inhibitoryactivity to an insect that is not inhibited by an otherwise insectinhibitory protein of the present invention to expand the spectrum ofinsect inhibition obtained, e.g., an additional polypeptide thatexhibits insect inhibitory activity to Hemipterans or Thysanopterans.

The possibility for insects to develop resistance to certaininsecticides has been documented in the art. One insect resistancemanagement strategy is to employ transgenic crops that express twodistinct insect inhibitory agents that operate through different modesof action. Therefore, any insects with resistance to either one of theinsect inhibitory agents can be controlled by the other insectinhibitory agent. Another insect resistance management strategy employsthe use of plants that are not protected to the targeted Coleopteran orLepidopteran pest species to provide a refuge for such unprotectedplants. One particular example is described in U.S. Pat. No. 6,551,962,which is incorporated by reference in its entirety.

Other embodiments such as topically applied pesticidal chemistries thatare designed for controlling pests that are also controlled by theproteins disclosed herein to be used with proteins in seed treatments,spray on, drip on, or wipe on formulations can be applied directly tothe soil (a soil drench), applied to growing plants expressing theproteins disclosed herein, or formulated to be applied to seedcontaining one or more transgenes encoding one or more of the proteinsdisclosed. Such formulations for use in seed treatments can be appliedwith various stickers and tackifiers known in the art. Such formulationscan contain pesticides that are synergistic in mode of action with theproteins disclosed, so that the formulation pesticides act through adifferent mode of action to control the same or similar pests that canbe controlled by the proteins disclosed, or that such pesticides act tocontrol pests within a broader host range or plant pest species that arenot effectively controlled by the TIC7040, TIC7040HT, TIC7042, TIC7381,TIC7382, TIC7383, TIC7386, TIC7388, TIC7389, variant, truncationvariant, chimera, or related family member pesticidal proteins.

The aforementioned composition/formulation can further comprise anagriculturally-acceptable carrier, such as a bait, a powder, dust,pellet, granule, spray, emulsion, a colloidal suspension, an aqueoussolution, a Bacillus spore/crystal preparation, a seed treatment, arecombinant plant cell/plant tissue/seed/plant transformed to expressone or more of the proteins, or bacterium transformed to express one ormore of the proteins. Depending on the level of insect inhibitory orinsecticidal inhibition inherent in the recombinant polypeptide and thelevel of formulation to be applied to a plant or diet assay, thecomposition/formulation can include various by weight amounts of therecombinant polypeptide, e.g. from 0.0001% to 0.001% to 0.01% to 1% to99% by weight of the recombinant polypeptide.

EXAMPLES

In view of the foregoing, those of skill in the art should appreciatethat changes can be made in the specific aspects which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention. Thus, disclosed specific structural andfunctional details are not to be interpreted as limiting. It should beunderstood that the entire disclosure of each cited reference isincorporated within this disclosure.

Example 1 Discovery of TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382,TIC7383, TIC7386, TIC7388, TIC7389

This Example describes the discovery of the pesticidal proteins TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, andTIC7389.

Sequences encoding novel Brevibacillus laterosporus (Bl) pesticidalproteins were identified, cloned, sequence confirmed and tested ininsect bioassay. The pesticidal proteins TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, TIC7383, TIC7386, TIC7388, and TIC7389 were isolatedfrom the Brevibacillus laterosporus strains listed in Table 3, andrepresent novel pesticidal proteins belonging to the Cry43 family oftoxins.

TABLE 3 Novel Cry43 pesticidal toxin proteins and correspondingBrevibacillus laterosporus strains. DNA Protein SEQ SEQ ID IDBrevibacillus Length Toxin NO: NO: laterosporus strains (a.a.) TIC7040 12 DSC005019 1259 TIC7040HT 3 4 DSC005019 1295 TIC7042 11 12 DSC0059731266 TIC7381 13 14 DSC006713 1269 TIC7382 15 16 DSC007657 1246 TIC738317 18 DSC008106 1256 TIC7386 29 30 DSC007651 1279 TIC7388 31 32DSC007962 1269 TIC7389 33 34 DSC006878 1319

Polymerase chain reaction (PCR) primers were designed based upon contigsderived from sequencing of each Brevibacillus laterosporus strainslisted in Table 3. Amplicons of the full length coding sequence for eachprotein toxin was produced using total DNA isolated from each strainlisted in Table 3. With respect to TIC7040, a coding sequence of 3,888bp was produced through amplification and differed from the predictedcoding sequence of 3,780 bp. The amplified coding sequence andcorresponding amino acid sequence were designated “TIC7040HT” todistinguish it from the original predicted contig, TIC7040. Each of theamplicons, with the exception of TIC7040, were cloned using methodsknown in the art into Bacillus thuringiensis (Bt) expression vectors inoperable linkage with a Bt expressible promoter.

Example 2 TIC7040HT, TIC7042, TIC7381, TIC7382, and TIC7383 DemonstrateColeopteran and Lepidopteran Activity in Insect Bioassay

This Example illustrates inhibitory activity exhibited by TIC7040HT,TIC7042, TIC7381, TIC7382, and TIC7383 proteins against various speciesof Coleoptera and Lepidoptera.

The pesticidal proteins TIC7040HT, TIC7042, TIC7381, TIC7382, andTIC7383 were expressed in Bt and assayed for toxicity against variousspecies of Lepidoptera and Coleoptera. Preparations of each toxin wereassayed against the Coleopteran species Western Corn Rootworm(Diabrotica virgifera virgifera, WCR), Cry3Bb-resistant Western CornRootworm (Diabrotica virgifera virgifera, WCRHP), Northern Corn Rootworm(Diabrotica barberi, NCR), Southern Corn Rootworm (Diabroticaundecimpunctata howardii, SCR), and Colorado potato beetle (Leptinotarsadecemlineata, CPB). Preparations of each toxin were also assayed againstthe Lepidopteran species Black Cutworm (Agrotis ipsilon, BCW), CornEarworm (Helicoverpa zea, (CEW), also known as Soybean Podworm andCotton Bollworm), Diamondback Moth (Plutella xylostella, DBM), EuropeanCorn Borer (Ostrinia nubilalis, ECB), Fall Armyworm (Spodopterafrugiperda, FAW), Southern Armyworm (Spodoptera eridania, SAW), SoybeanLooper (Pseudoplusia includes, SBL), Southwestern Corn Borer (Diatraeagrandiosella, SWCB), tobacco budworm (Heliothis virescens, TBW), andVelvetbean Caterpillar (Anticarsia gemmatalis, VBC). The toxinpreparations were also assayed against the Hemipteran species Tarnishedplant bug (Lygus lineolaris), Western tarnished plant bug (Lygushesperus), and Neotropical Brown Stink Bug (Euschistus heros).

Transformed Bt expressing TIC7040HT, TIC7042, TIC7381, TIC7382, orTIC7383 were grown for twenty four (24) hours, and spores as well assolubilized proteins were added to the insect diet for assay. Mortalityand stunting were evaluated by comparing the growth and development ofinsects on a diet with a culture from a Bt strain expressing TIC7040HT,TIC7042, TIC7381, TIC7382, or TIC7383 to insects on a diet with anuntreated control culture. Activity was not observed for Hemipteraninsect pests for TIC7040HT, TIC7042, TIC7381, TIC7382, or TIC7383.Activity was observed for Coleopteran and Lepidopteran insect pests. Thebioassay activity with respect to stunting (S) and mortality (M)observed for each protein is presented in Tables 4 (Coleoptera) and 5(Lepidoptera), wherein “+” indicates activity, an empty cell indicatesno activity observed, and “NT” indicates the toxin was not assayedagainst that specific insect pest.

TABLE 4 Bioassay activity of TIC7040HT, TIC7042, TIC7381, TIC7382, andTIC7383 against Coleopteran insect pests. WCR WCRHP NCR SCR CPB Toxin SM S M S M S M S M TIC7040HT + + + + + + + + + TIC7042 + + + + + + + + +TIC7381 + + + + + + + + + + TIC7382 + + + + + + + + +TIC7383 + + + + + + + + + TIC7389 + + NT NT NT NT NT NT + +

TABLE 5 Bioassay activity of TIC7040HT, TIC7042, TIC7381, TIC7382, andTIC7383 against Lepidopteran insect pests. BCW CEW DBM ECB FAW SAW SBLSWCB TBW VBC Toxin S M S M S M S M S M S M S M S M S M S MTIC7040HT + + + + + + + + + TIC7042 + + + + + + + +TIC7381 + + + + + + + + + + TIC7382 + + + + + + + + TIC7383 + + + + +

As can be seen in Tables 4 and 5, the pesticidal proteins TIC7040HT,TIC7042, TIC7381, TIC7382, and TIC7383 demonstrated activity againstmany of the Coleopteran and Lepidopteran insect pest species. TIC7040HTdemonstrated activity against the Coleopteran pests WCR, WCRHP, NCR,SCR, and CPB; and the Lepidopteran insect pests CEW, DBM, ECB, FAW, SBL,SWCB, and VBC. TIC7042 demonstrated activity against the Coleopteranpests WCR, WCRHP, NCR, SCR, and CPB; and the Lepidopteran insect pestsBCW, CEW, DBM, ECB, FAW, SBL, and VBC. Mortality caused by TIC7042against WCR was very high. TIC7381 demonstrated activity against theColeopteran pests WCR, WCRHP, NCR, SCR, and CPB; and the Lepidopteraninsect pests CEW, DBM, ECB, FAW, SBL, SWCB, and VBC. TIC7382demonstrated activity against the Coleopteran pests WCR, WCRHP, NCR,SCR, and CPB; and the Lepidopteran insect pests DBM, ECB, SBL, SWCB, andVBC. TIC7383 demonstrated activity against the Coleopteran pests WCR,WCRHP, NCR, SCR, and CPB and; the Lepidopteran insect pests CEW, DBM,ECB, and VBC.

The pesticidal protein TIC7389 demonstrated activity against theColeopteran pests, WCR and CPB. TIC7389 was also assayed against theLepidopteran insect pest species Corn Earworm, Fall Armyworm, andSoybean Looper, as well as the Hemipteran species Tarnished plant bugand Western tarnished plant bug. Stunting was observed for SBL, but notthe other two Lepidopteran species and the two Hemipteran insect pests.

The insect toxins TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, andTIC7389 demonstrate activity against a variety of Coleopteran andLepidopteran insect pest species. The insect toxin TIC7389 demonstratestoxicity to the Lepidopteran insect pest Soybean Looper.

Example 3 Tryptic Digested TIC7040HT and TIC7383, and ChymotrypticDigested TIC7040HT Demonstrate Activity Against Western Corn Rootworm

This Example illustrates inhibitory activity exhibited by tryptic andchymotryptic digested TIC7040HT, and tryptic digested TIC7383 proteinsagainst Western Corn Rootworm.

Protein samples of TIC7040HT were subjected to tryptic and chymotrypticdigest in separate reactions. Likewise, TIC7383 was subjected to atryptic digest. The tryptic and chymotryptic digests were performedusing methods known in the art. The digested proteins were analyzed bymass spectrometry to determine the resulting protein fragments. Theresulting tryptic and chymotryptic fragments of TIC7040HT, and theresulting tryptic fragment of TIC7383 are presented in Table 6.

The digested proteins were used in bioassay against Western CornRootworm. Table 6 shows the assay of activity for each digested proteinpreparation, and the full length TIC7040HT and TIC7383 proteins.

TABLE 6 Bioassay activity of TIC7040HT, tryptic and chymotrypticdigested TIC7040HT, TIC7383, and tryptic digested TIC7383 againstWestern Corn Rootworm. Amino Acid Position Relative DNA Protein to FullSEQ ID SEQ ID Length Toxin NO: NO: Protein Stunting Mortality TIC7040HT3 4 + + TIC7040HT_Tryp 70 71 43-624 + + TIC7040HT_Chymo 72 73 45-641 + +TIC7383 17 18 + + TIC7383_Tryp 74 75 55-668 + +

As can be seen in Table 6, the tryptic and chymotryptic digestedTIC7040HT and the tryptic digested TIC7383 proteins retained activityagainst Western Corn Rootworm.

Example 4 Truncations of TIC7040HT and TIC7383 Demonstrate ColeopteranActivity in Insect Bioassay

This Example illustrates inhibitory activity exhibited by truncations ofthe TIC7040HT and TIC7383 proteins against various species ofColeoptera.

Coding sequences encoding truncations of TIC7040HT and TIC7383 wereproduced using methods known in the art and cloned into bacterialexpression vectors to be expressed in Bt. The truncated proteins wereprovided in insect diets and assayed for activity against Coleopteraninsect pests. The amino acid positions of the truncated TIC7040HT andTIC7383 toxins relative to the full length TIC7040HT and TIC7383 areshown in Table 7.

Transformed Bt expressing truncations of TIC7040HT and TIC7383 weregrown for twenty four (24) hours and spores, as well as solubilizedproteins, were added to insect diet for assay. Mortality and stuntingwere evaluated by comparing the growth and development of insects on adiet with a culture from the Bt strain expressing truncations ofTIC7040HT or TIC7383 to insects on a diet with an untreated controlculture.

The truncations of TIC7040HT and TIC7383 were assayed for toxicityagainst various species of Coleoptera. Preparations of each toxin wereassayed against the Coleopteran species Western Corn Rootworm,Cry3Bb-resistant Western Corn Rootworm, and Colorado potato beetle.

Activity was observed for Coleopteran insect pests. The bioassayactivity with respect to stunting (S) and mortality (M) observed foreach protein is presented in Table 7, wherein “+” indicates activity, anempty cell indicates no activity observed, and “NT” indicates the toxinwas not assayed against that specific insect pest. The activity of thefull length TIC7040HT and TIC7383 is also provided for comparison to thetruncated protein activity.

TABLE 7 Bioassay activity of truncations of TIC7040HT and TIC7383against Coleopteran insect pests. Amino Acid Positions DNA ProteinRelative to SEQ SEQ Full ID ID Length WCR WCRHP NCR SCR CPB Toxin NO:NO: Protein S M S M S M S M S M TIC7040HT 3 4 + + + + + + + + +TIC7040HT_5 7 8 13-611 + + NT NT NT NT + + TIC7040HT_6 9 10 13-671 + +NT NT NT NT + + TIC7383 17 18 + + + + + + + + + TIC7383_2 19 2012-1256 + + NT NT NT NT + + TIC7383_3 21 22  1-659 + + + NT NT NT NT + +TIC7383_4 23 24  1-679 + + NT NT NT NT + + TIC7383_5 25 26 15-659 + + NTNT NT NT + + TIC7383_6 27 28 15-679 + + NT NT NT NT + +

As can be seen in Table 7, truncations of TIC7040HT and TIC7383maintained activity against WCR and CPB.

Example 5 Design of Synthetic Coding Sequences Encoding TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, and Truncation Variantsof TIC7040HT, TIC7042, TIC7382, and TIC7383 for Expression in PlantCells

Synthetic or artificial coding sequences were constructed for use inexpression of TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383,and truncation variants of TIC7040HT, TIC7042, TIC7382, and TIC7383, inplants. These synthetic coding sequences were cloned into a binary planttransformation vectors, and used to transform plant cells. The syntheticnucleic acid sequences were synthesized according to methods generallydescribed in U.S. Pat. No. 5,500,365, avoiding certain inimical problemsequences such as ATTTA and A/T rich plant polyadenylation sequenceswhile preserving the amino acid sequence of the native Bl protein. Thesynthetic coding sequences for the TIC7040, TIC7040HT, TIC7042, TIC7381,TIC7382, TIC7383, and truncation variants of TIC7040HT, TIC7042,TIC7382, and TIC7383 pesticidal proteins are presented Table 8.

TABLE 8 Synthetic coding sequences used for expression in plant cellsencoding TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, andtruncation variants of TIC7040HT, TIC7042, TIC7382, and TIC7383.Additional Amino Acid Alanine Position DNA Protein Residue Relative toSEQ SEQ after Full Length ID ID Initiating N-terminal C-terminal Proteinand Description NO: NO: Methionine Truncation Truncation MutationsCR-BREla.TIC7040.nno_Mc:1 35 2 No No No CR-BREla.TIC7040_10.nno_Mc:1 364 No No No CR-BREla.TIC7040_10.nno_Mc:3 37 4 No No NoCR-BREla.TIC7040_10.nno_Mc:4 38 4 No No No CR-BREla.TIC7040_10.nno_Mc:539 4 No No No CR-BREla.TIC7040_10.nno_Mc:6 40 4 No No NoCR-BREla.TIC7040_10.nno_Mc:7 41 4 No No No CR-BREla.TIC7040_1.nno_Mc:142 43 No Yes Yes 15-651 CR-BREla.TIC7040_2.nno_Mc:1 44 10 No Yes Yes13-671 CR-BREla.TIC7040_11.nno_Mc:1 45 46 No Yes Yes 14-671CR-BREla.TIC7040_12.nno_Mc:2 47 48 No No Yes  1-660CR-BREla.TIC7040_13.nno_Mc:1 49 50 No No Yes  1-627CR-BREla.TIC7040_14.nno_Mc:1 76 77 Yes Yes Yes 52-660CR-BREla.TIC7042.nno_Mc:1 51 12 No No No CR-BREla.TIC7042_1.nno_Mc:1 5253 No Yes Yes 11-646 CR-BREla.TIC7042_2.nno_Mc:1 54 55 No Yes Yes 11-665CR-BREla.TIC7381_1.nno_Mc:1 56 57 Yes No No CR-BREla.TIC7381_2.nno_Mc:178 79 Yes No Yes  1-658 CR-BREla.TIC7381_3.nno_Mc:1 80 81 Yes Yes Yes50-658 CR-BREla.TIC7382_1.nno_Mc:1 58 59 Yes No NoCR-BREla.TIC7382_2.nno_Mc:1 60 61 Yes No Yes  1-659CR-BREla.TIC7382_3.nno_Mc:1 82 83 Yes Yes Yes 52-659CR-BREla.TIC7383_1.nno_Mc:1 62 63 Yes No No CR-BREla.TIC7383_7.nno_Mc:164 65 Yes Yes Yes 54-668 CR-BREla.TIC7383_8.nno_Mc:1 66 67 Yes No Yes 1-661 CR-BREla.TIC7383_9.nno_Mc:1 68 69 Yes No Yes  1-668CR-BREla.TIC7383_19.nno_Mc:1 84 85 No Yes Yes 15-668CR-BREla.TIC7383_20.nno_Mc:1 86 87 No Yes Yes 15-661CR-BREla.TIC7383_21.nno_Mc:1 88 89 Yes Yes Yes 54-661CR-BREla.TIC7383_22.nno_Mc:1 90 91 No Yes Yes 54-668CR-BREla.TIC7383_23.nno_Mc:1 92 93 No Yes Yes 54-661CR-BREla.TIC7383_24.nno_Mc:2 94 95 No Yes Yes 73-661CR-BREla.TIC7383_25.nno_Mc:3 96 97 No Yes Yes 94-661CR-BREla.TIC7383_26.nno_Mc:1 98 99 No Yes Yes 114-661 CR-BREla.TIC7383_27.nno_Mc:1 100 101 Yes Yes Yes 54-658CR-BREla.TIC7383_28.nno_Mc:1 102 103 No Yes Yes 15-658CR-BREla.TIC7383_29.nno_Mc:1 104 105 Yes No Yes 1-963CR-BREla.TIC7383_30.nno_Mc:1 106 107 Yes No No K964A; R966A; K968ACR-BREla.TIC7383_31.nno_Mc:1 108 109 Yes No Yes 1-1065; K964A; R966A;K968A CR-BREla.TIC7383_32.nno_Mc:1 110 111 Yes No No Deletion 964-969

Example 6 Expression Cassettes for Expression of TIC7040, TIC7040HT,TIC7042, TIC7381, TIC7382, TIC7383, and Truncation Variants ofTIC7040HT, TIC7042, TIC7382, and TIC7383 in Plant Cells

A variety of plant expression cassettes were designed with the sequencesas set forth in Table 8. Such expression cassettes are useful fortransient expression in plant protoplasts or transformation of plantcells. Typical expression cassettes are designed with respect to theeventual placement of the protein within the plant cell. For a plastidtargeted protein, the synthetic TIC7040, TIC7040HT, TIC7042, TIC7381,TIC7382, TIC7383 or truncation variant of TIC7040HT, TIC7042, TIC7382,or TIC7383 pesticidal protein coding sequences are operably linked inframe with a chloroplast targeting signal peptide coding sequence. Theresulting plant transformation vectors comprise a first transgenecassette for expression of the pesticidal protein which comprises aconstitutive promoter, operably linked 5′ to a leader, operably linked5′ to an intron (or optionally no intron), operably linked 5′ to asynthetic coding sequence encoding a plastid targeted or untargetedTIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, or truncationvariant of TIC7040HT, TIC7042, TIC7382, or TIC7383 protein, which is inturn operably linked 5′ to a 3′ UTR and, a second transgene cassette forthe selection of transformed plant cells using glyphosate or antibioticselection. All of the elements described above are arranged contiguouslyoften with additional sequence provided for the construction of theexpression cassette such as restriction endonuclease sites or ligationindependent cloning sites.

Example 7 TIC7382 Provides Efficacious Resistance to Western CornRootworm when Expressed in Stably Transformed Corn Plants

This Example illustrates the inhibitory activity exhibited bytruncations of TIC7382 against Coleoptera, such as Western CornRootworm, when expressed in plants and provided as a diet to therespective insect pest.

Binary plant transformation vectors comprising transgene cassettesdesigned to express untargeted TIC7382 (CR-BRE1a.TIC7382_1.nno_Mc:1) andthe truncation variants CR-BRE1a.TIC7382_2.nno_Mc:1 andCR-BRE1a.TIC7382_3.nno_Mc:1 were cloned using methods known in the art.The plant transformation vectors comprised a first transgene cassettefor expression of the TIC7382 pesticidal protein or one of thetruncation variants which comprised a root preferred promoter, operablylinked 5′ to a leader, operably linked 5′ to an intron, operably linked5′ to a synthetic coding sequence encoding TIC7382 or the truncationvariants, each of which comprised an additional alanine residueimmediately following the initiating methionine, which was in turn wasoperably linked 5′ to a 3′ UTR and, a second transgene cassette for theselection of transformed plant cells using glyphosate. The resultingvectors were used to stably transform corn plants using methods known inthe art. Single T-DNA insertion events were selected and grown.Pesticidal activity was assayed against Western Corn Rootworm feeding onthe roots of the stably transformed corn plants.

R₀ stably transformed plants were used to assay for Coleopteranresistance as well as generating F₁ progeny. Multiple single copy eventswere selected from each binary vector transformation. A portion of thoseevents arising from each binary vector transformation were used in theColeopteran assay, while another portion of events were used to generateF₁ progeny for further testing.

The R₀ assay plants were transplanted to eight inch pots. The plantswere inoculated with eggs from Western Corn Rootworm. The eggs wereincubated for approximately ten (10) days prior to inoculation to allowhatching to occur four (4) days after inoculation to ensure a sufficientnumber of larvae survive and are able to attack the corn roots. Thetransformed plants were inoculated at approximately V2 to V3 stage. Theplants were grown after infestation for approximately twenty eight (28)days. The plants were removed from the pots with the roots beingcarefully washed to remove all soil. The damage to the roots wasassessed using a damage rating scale of 1-5, as presented in Table 9.Comparison was also made to the negative control to assure the assay hasbeen performed properly. Low root damage scores indicate resistanceconferred by the TIC7382 protein or the truncation variants to WCR. AnRDR score of 1.0 to 2.5 represents good efficacy, an RDR score of 2.6 to3.5 represents medium efficacy, and an RDR score of 3.6 to 5.0represents low efficacy.

TABLE 9 R₀ root damage rating scores. Root Damage Score Description 1 Novisible feeding 2 Some feeding; no pruning 3 Pruning of at least oneroot 4 Entire node pruned 5 More than one node pruned

For F₁ assay, eggs from Western Corn Rootworm were incubated forapproximately ten (10) days to allow hatching within four (4) days afterinoculation. The plants were inoculated at approximately V2 to V3 stage.Each pot was inoculated with about two thousand eggs. The plants weregrown after infestation for approximately twenty eight (28) days. Theplants were removed from the pots with the roots being carefully washedto remove all soil. The damage to the roots were assessed using a damagerating scale of 0-3, as presented in Table 10. Comparison was made tothe negative control to assure the assay was performed properly. Lowroot damage scores indicate resistance conferred TIC7382, or thetruncation to WCR. An RDR at F₁ of 0.0 to 0.75 represents good efficacy,an RDR of 0.76 to 1.5 represents medium efficacy, and an RDR of 1.6 to3.0 represents low efficacy.

TABLE 10 F₁ root damage rating scores. Root Damage Score Description 0No visible feeding 0.01-0.09 Feeding scars and tracks 0.1-0.9 Rootpruning, but less than a full node 1.0-1.9 At least a full node (orequivalent) destroyed to within 1.5 inches of plant 2.0-2.9 Two or morenodes gone 3 Three or more nodes gone

Table 11 shows the average Root Damage Rating (RDR) assayed for theTIC7382 protein and truncation variants.

TABLE 11 Average Root Damage Rating (RDR) of transgenic corn plantsexpressing TIC7382 or truncation variants. Amino Acid Position CodingProtein Relative Sequence SEQ to Full SEQ ID ID Length Average AverageDescription NO: NO: Protein R₀ RDR F₁ RDR CR-BREla.TIC7382_ 58 59 3.72.4 1.nno_Mc:1 CR-BREla.TIC7382_ 60 61  1-658 3.3 1.1 2.nno_Mc:1CR-BREla.TIC7382_ 82 83 50-658 1.6 1.0 3.nno_Mc:1

As can be seen in Table 11, a C-terminal truncation of the TIC7382protein giving rise to the truncation variant,CR-BRE1a.TIC7382_2.nno_Mc:1, improved efficacy relative to the RootDamage Ratings of CR-BRE1a.TIC7382_1.nno_Mc:1. Truncation of the TIC7382protein at both the N-terminus and C-terminus giving rise to thetruncation variant, CR-BRE1a.TIC7382_3.nno_Mc:1, resulted in less damageto the corn roots and further improved efficacy as demonstrated by alower R₀ Root Damage Rating when compared to the Root Damage Ratings ofCR-BRE1a.TIC7382_1.nno_Mc:1 and CR-BRE1a.TIC7382_2.nno_Mc: 1.

Example 8 Assay of Activity of TIC7040, TIC7040HT, TIC7042, TIC7381,TIC7382, TIC7383, and Truncation Variants of TIC7040HT, TIC7042,TIC7382, or TIC7383 Against Coleopteran Corn Rootworm Pests whenExpressed in Stably Transformed Corn Plants

This Example illustrates the inhibitory activity of TIC7040, TIC7040HT,TIC7042, TIC7381, TIC7382, TIC7383, or truncation variants of TIC7040HT,TIC7042, TIC7382, or TIC7383 against different Coleopteran species thatfeed on corn roots.

Binary plant transformation vectors comprising transgene cassettesdesigned to express both plastid targeted and untargeted TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, or truncation variants ofTIC7040HT, TIC7042, TIC7382, or TIC7383 are cloned using methods knownin the art and comprise the sequences as shown in Table 8. The resultingvectors are used to stably transform corn plants using methods known inthe art. Single T-DNA insertion events are selected and grown.Pesticidal activity is assayed against the Coleopteran pests WesternCorn Rootworm (Diabrotica virgifera virgifera, WCR), Northern CornRootworm (Diabrotica barberi, NCR), Mexican Corn Rootworm (Diabroticavirgifera zeae, MCR), Brazilian Corn Rootworm (Diabrotica balteata,BZR), Southern Corn Rootworm (Diabrotica undecimpunctata howardii, SCR),Colorado potato beetle (Leptinotarsa decemlineata, CPB), or a BrazilianCorn Rootworm complex (BCR, consisting of Diabrotica viridula andDiabrotica speciosa) feeding on the roots of the stably transformed cornplants.

R₀ stably transformed plants are used to assay for Coleopteranresistance as well as generating F₁ progeny. Multiple single copy eventsare selected from each binary vector transformation. A portion of theevents arising from each binary vector transformation are used in the R₀Coleopteran assay, while another portion of events are used to generateF₁ progeny for further testing.

The R₀ assay plants are transplanted to eight inch pots. The plants areinoculated with eggs from Western Corn Rootworm, Northern Corn Rootworm,or Southern Corn Rootworm. The eggs are incubated for approximately ten(10) days prior to inoculation to allow hatching to occur four (4) daysafter inoculation to ensure a sufficient number of larvae survive andare able to attack the corn roots. The transformed plants are inoculatedat approximately V2 to V3 stage. The plants are grown after infestationfor approximately twenty eight (28) days. The plants are removed fromthe pots with the roots being carefully washed to remove all soil. Thedamage to the roots is assessed using a damage rating scale of 1-5, aspresented in Table 9 in Example 5. Comparison is also made to thenegative controls to assure the assay has been performed properly.Multiple R₀ events for each binary vector transformation are used in theColeopteran assay. Low root damage scores indicate resistance conferredby TIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, or truncationvariants of TIC7040HT, TIC7042, TIC7382, or TIC7383 to the testedColeopteran pest.

A portion of the R₀ stably transformed events arising from each binaryvector transformation are used to produce F₁ progeny. The R₀ stablytransformed plants are allowed to self-fertilize, producing F₁ progeny.The F₁ seed is planted. Heterozygous plants are identified throughmolecular methods known in the art and used for assay againstColeopteran pests, as well as ELISA expression measurements of toxinprotein. A portion of the heterozygous F₁ progeny from each event areused for insect assay, while another portion is used to measure toxinprotein expression.

Eggs from Western Corn Rootworm, Northern Corn Rootworm, or SouthernCorn Rootworm are incubated for approximately ten (10) days to allowhatching within four (4) days after inoculation. For WCR, each pot isinoculated with about two thousand eggs. For NCR, less eggs may be useddue to the lower availability of eggs from this species. The plants areinoculated at approximately V2 to V3 stage. The plants are grown afterinfestation for approximately twenty eight (28) days. The plants areremoved from the pots with the roots being carefully washed to removeall soil. The damage to the roots are assessed using a damage ratingscale of 0-3, as presented in Table 10 in Example 5. Comparison is madeto the negative control to assure the assay has been performed properly.Low root damage scores indicate resistance conferred by TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, or truncation variants ofTIC7040HT, TIC7042, TIC7382, or TIC7383 protein to the Coleopteran pest.

Example 9 Assay of Activity of TIC7040, TIC7040HT, TIC7042, TIC7381,TIC7382, TIC7383, or Truncation Variants of TIC7040HT, TIC7042, TIC7382,or TIC7383 Against Lepidopteran Pests when Expressed in StablyTransformed Corn, Soybean, or Cotton Plants

This Example illustrates the assay of activity against variousLepidopteran pest species fed tissue from stably transformed corn,soybean or cotton plants expressing TIC7040, TIC7040HT, TIC7042,TIC7381, TIC7382, TIC7383, or truncation variants of TIC7040HT, TIC7042,TIC7382, or TIC7383.

Binary plant transformation vectors comprising transgene cassettesdesigned to express both plastid targeted and untargeted TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, or truncation variants ofTIC7040HT, TIC7042, TIC7382, or TIC7383 are cloned using methods knownin the art and comprise the coding sequences as presented in Table 8.

Corn, soybean, or cotton is transformed with the binary transformationvectors described above using an Agrobacterium-mediated transformationmethod. The transformed cells are induced to form plants by methodsknown in the art. Bioassays using plant leaf disks are performedanalogous to those described in U.S. Pat. No. 8,344,207. Anon-transformed corn, soybean, or cotton plant is used to obtain tissueto be used as a negative control. Multiple transformation events fromeach binary vector are assessed against Lepidopteran pests such as, butnot limited to, Black Cutworm, Corn Earworm, Diamondback Moth, EuropeanCorn Borer, Fall Armyworm, Southern Armyworm, Soybean Looper,Southwestern Corn Borer, Tobacco budworm, and Velvetbean Caterpillar.Those insects demonstrating stunting and/or mortality in the insectbioassay are determined to be susceptible to the effects of the testedTIC7040, TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, or truncationvariant of TIC7040HT, TIC7042, TIC7382, or TIC7383 insect toxinpesticidal protein.

Example 10 Assay of Activity of TIC7040, TIC7040HT, TIC7042, TIC7381,TIC7382, TIC7383, or Truncation Variants of TIC7040HT, TIC7042, TIC7382,or TIC7383 Against Flea Beetle Pests when Expressed in StablyTransformed Canola Plants

This Example illustrates the assay of activity against various speciesof Flea Beetle when allowed to feed on whole transgenic canola plants ortissues derived from transgenic canola plants expressing TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, or truncation variants ofTIC7040HT, TIC7042, TIC7382, or TIC7383.

Binary plant transformation vectors comprising transgene cassettesdesigned to express both plastid targeted and untargeted TIC7040,TIC7040HT, TIC7042, TIC7381, TIC7382, TIC7383, or truncation variants ofTIC7040HT, TIC7042, TIC7382, or TIC7383 are cloned using methods knownin the art and comprise the coding sequences as presented in Table 8.

The resulting binary transformation vectors are used to stably transformcanola plant cells using methods known in the art. The transformed cellsare induced to form plants. Bioassays using plant leaf disks areperformed analogous to those described in U.S. Pat. No. 8,344,207 usingfield collected Flea Beetles. A non-transformed canola plant is used toobtain tissue to be used as a negative control. Multiple transformationevents from each binary vector are assessed against Coleopteran FleaBeetle pests such as, but not limited to, Crucifer Flea Beetle(Phyllotreta cruciferae), Striped Flea Beetle (Phyllotreta striolata),and Western Black Flea Beetle (Phyllotreta pusilla). Flea Beetlemortality is determined each day as the Beetles continue to feed. Leafdiscs are changed every two (2) to three (3) days over a twelve (12) dayperiod to assure fresh material is available to the Flea Beetles forfeeding, and to reduce any impact of protein degradation in the sample.

Alternatively, transformed canola plants can be planted in a field whereFlea Beetle infestations are present. The plants can be housed in a tentto prevent those Flea Beetles that emerge from the soil from escapingthe experimental plots. Damage ratings of the canola leaves can be takento determine which plants experienced lesser damage and demonstratedresistance to the Flea Beetles.

Example 11 Truncation of TIC7383 Improves Efficacy Against Corn Rootwormin Stably Transformed Corn Plants

This Example illustrates the improvement of efficacy of the TIC7383through truncations at the N-terminus, the C-terminus, or both termini.

Binary plant transformation vectors comprising transgene cassettesdesigned to express untargeted TIC7383 and truncation variants werecloned using method known in the art. The plant transformation vectorscomprised a first transgene cassette for expression of the TIC7383pesticidal protein or the truncation variants which comprised a rootpreferred promoter, operably linked 5′ to a leader, operably linked 5′to an intron, operably linked 5′ to a synthetic coding sequence encodingTIC7383 or the truncation variants, some of which comprised anadditional alanine residue immediately following the initiatingmethionine, which was in turn was operably linked 5′ to a 3′ UTR and, asecond transgene cassette for the selection of transformed plant cellsusing glyphosate. The resulting vectors were used to stably transformcorn plants using methods known in the art. Single T-DNA insertionevents were selected and grown. Pesticidal activity was assayed againstWestern Corn Rootworm feeding on the roots of the stably transformedcorn plants.

R₀ stably transformed plants were used to assay for resistance toWestern Corn Rootworm as well as generating F₁ progeny. Multiple singlecopy events were selected from each binary vector transformation. Aportion of those events arising from each binary vector transformationwere used in the Coleopteran assay, while another portion of events wereused to generate F₁ progeny for further testing. R₀ and F₁ Root DamageRating scores were determined using the Root Damage Rating scores aspresented in Tables 9 and 10, respectively, presented in Example 7.Table 12 shows the average Root Damage Rating scores obtained from theR₀ and F₁ stably transformed corn plants expressing TIC7383 andtruncation variants. Protein expression levels of TIC7383 and thecorresponding truncation variants are also shown in Table 12 and areexpressed as parts per million (ppm).

As can be seen in Table 12, truncations at the N-terminus, C-terminus,or both termini improved efficacy in some of the truncation variants asdemonstrated by lower average Root Damage Rating scores relative to thefull length TIC7383 protein (CR-BRE1a.TIC7383_1.nno_Mc:1). Truncationsof the TIC7383 toxin also improved expression within the plant in mostinstances as well.

TABLE 12 Average Root Damage Rating (RDR) scores of stably transformedcorn plants expressing TIC7383 and truncation variants. Amino AcidAdditional Position Alanine Relative Residue to CR- after BREla. ProteinInitiating TIC7383_1. Expression Average Average Description Methioninenno_Mc:1 (ppm) R₀ RDR F₁ RDR CR-BREla. Yes 11.9 3.8 2.2 TIC7383_1.nno_Mc:1 CR-BREla. Yes  54-668 421.3 2.9 1.6 TIC7383_ 7.nno_Mc:1CR-BREla. Yes  1-661 289.2 2.4 TIC7383_ 8.nno_Mc:1 CR-BREla. Yes  1-668317.2 3.3 TIC7383_ 9.nno_Mc:1 CR-BREla. No  15-668 23.7 3.3 TIC7383_19.nno_Mc:1 CR-BREla. No  15-661 29.1 3.9 TIC7383_ 20.nno_Mc:1 CR-BREla.Yes  54-661 617.4 2.7 TIC7383_ 21.nno_Mc:1 CR-BREla. No  54-668 451.32.6 TIC7383_ 22.nno_Mc:1 CR-BREla. No  54-661 343.1 2.8 TIC7383_23.nno_Mc:1 CR-BREla. No  73-661 356.5 2.9 TIC7383_ 24.nno_Mc:2CR-BREla. No 114-661 8.5 3.9 TIC7383_ 26.nno_Mc:1 CR-BREla. Yes  54-658356.2 2.9 TIC7383_ 27.nno_Mc:1 CR-BREla. No  15-658 25.4 3.7 TIC7383_28.nno_Mc:1

Example 12 Assay of Activity of Chimeras of TIC7381, TIC7382, TIC7383and TIC7042 Against Western Corn Rootworm in Stably Transformed CornPlants

This Example illustrates the design of chimeras of TIC7381, TIC7382,TIC7383, and TIC7042 and the assay of activity against Western CornRootworm (WCR) in stably transformed corn plants expressing thechimeras.

Chimeras of TIC7381, TIC7382, TIC7383, and TIC7042 were designed whereindomains one and two (D1D2) of one toxin were combined with the thirddomain (D3) of another toxin. Table 13 below shows the composition ofeach chimera.

TABLE 13 Composition of the TIC7381, TIC7382, TIC7383, and TIC7042chimeras. Nucleotide Protein SEQ SEQ Description ID NO: ID NO: D1D2 D3GOI-TIC10743.nno_Mc:1 112 113 TIC7383 TIC7042 GOI-TIC10744.nno_Mc:1 114115 TIC7383 TIC7381 GOI-TIC10745.nno_Mc:1 116 117 TIC7383 TIC7382GOI-TIC10746.nno_Mc:1 118 119 TIC7382 TIC7383 GOI-TIC10747.nno_Mc:1 120121 TIC7381 TIC7383 GOI-TIC10748.nno_Mc:1 122 123 TIC7042 TIC7383

Binary plant transformation vectors comprising transgene cassettesdesigned to express untargeted chimeras of TIC7381, TIC7382, TIC7383,and TIC7042 were cloned using methods known in the art. The planttransformation vectors comprised a first transgene cassette forexpression of the chimeric toxin pesticidal protein which comprised aroot preferred promoter, operably linked 5′ to a leader, operably linked5′ to an intron, operably linked 5′ to a synthetic coding sequenceencoding the chimeras of TIC7381, TIC7382, TIC7383, and TIC7042, whichcomprised an additional alanine residue immediately following theinitiating methionine, which was in turn was operably linked 5′ to a 3′UTR and, a second transgene cassette for the selection of transformedplant cells using glyphosate. The resulting vectors were used to stablytransform corn plants using methods known in the art. Single T-DNAinsertion events were selected and grown. Pesticidal activity wasassayed against Western Corn Rootworm feeding on the roots of the stablytransformed corn plants.

R₀ stably transformed plants were used to assay for resistance toWestern Corn Rootworm as well as generating F₁ progeny. Multiple singlecopy events were selected from each binary vector transformation. Aportion of those events arising from each binary vector transformationwere used in the Coleopteran assay, while another portion of events wereused to generate F₁ progeny for further testing. R₀ and F₁ Root DamageRating scores were determined using the Root Damage Rating scores aspresented in Tables 9 and 10, respectively, presented in Example 7.Table 14 shows the average Root Damage Rating scores obtained from theR₀ and F₁ stably transformed corn plants expressing the chimeras ofTIC7381, TIC7382, TIC7383, and TIC7042 or truncation variants, wherein“NT” indicates not tested. Comparison is made to a TIC7382 varianttruncated at the N-terminus and C-terminus protoxin domain(CR-BRE1a.TIC7382_3.nno_Mc:1) and a TIC7383 variant truncated at theN-terminus and C-terminus protoxin domain (CR-BRE1a.TIC7383_7.nno_Mc:1).

TABLE 14 Average Root Damage Rating (RDR) scores of stably transformedcorn plants expressing chimeras of TIC7381, TIC7382, TIC7383, andTIC7042 against Western Corn Rootworm. Average R₀ Average F₁ DescriptionRDR RDR Negative Control 2.8 GOI-TIC10743.nno_Mc:1 3.4 2GOI-TIC10744.nno_Mc:1 3.3 2.3 GOI-TIC10745.nno_Mc:1 3.8 2.6GOI-TIC10746.nno_Mc:1 1.3 0.4 GOI-TIC10747.nno_Mc:1 NT NTGOI-TIC10748.nno_Mc:1 3.3 2.7 CR-BREla.TIC7382_3.nno_Mc:1 1.6 1.0CR-BREla.TIC7383_7.nno_Mc:1 3 1.5

As can be seen in Table 14, the chimeric toxin GOI-TIC10746.nno_Mc:1comprised of domains one and two of TIC7382 and domain three of TIC7383gave better efficacy at R₀ and F₁ relative to the negative control,CR-BRE1a.TIC7382_3.nno_Mc:1, and CR-BRE1a.TIC7383_7.nno_Mc: 1. Four F₁events expressing GOI-TIC10746.nno_Mc:1 were included in the assay. RootDamage Ratings scores ranged from 0.11 to 0.79 with (RDR: 0.11, 0.20,0.30, and 0.79). The one GOI-TIC10746.nno_Mc:1 expressing event with anRDR of 0.11 was equal to a commercial control. In addition to the datapresented above, protein isolated from bacteria expressing the chimerictoxin GOI-TIC10746.nno_Mc:1 also demonstrated activity against NorthernCorn Rootworm when presented in a diet bioassay.

Example 13 Assay of Activity of Chimeras of TIC7382 and TIC7383 CornRootworm in Stably Transformed Corn Plants

This Example illustrates the design of chimeras of TIC7382 and TIC7383and the assay of activity against Western Corn Rootworm (WCR) in stablytransformed corn plants expressing the chimeras.

Synthetic coding sequences encoding chimeric toxins comprised of domainsone and two of TIC7382 and domain three of TIC7383 designed forexpression in plants are cloned using methods known in the art. Thechimeric toxin, TIC10746NTermExt1 (SEQ ID NO:125, encoded by SEQ IDNO:124) comprises the N-terminal extension of TIC7382. The chimerictoxin, TIC10746NTermExt2 (SEQ ID NO:127, encoded by SEQ ID NO:126)comprises the N-terminal extension of TIC7383. The synthetic codingsequences are cloned in binary plant transformation vectors similar tothose described in Example 12 and used to transform corn plants. Rootassays are performed as previously described in Example 8 for R₀ and F₁plants after infestation of such corn rootworm species as Western CornRootworm (Diabrotica virgifera virgifera, WCR), Northern Corn Rootworm(Diabrotica barberi, NCR), Mexican Corn Rootworm (Diabrotica virgiferazeae, MCR), Brazilian Corn Rootworm (Diabrotica balteata, BZR), SouthernCorn Rootworm (Diabrotica undecimpunctata howardii, SCR), Coloradopotato beetle (Leptinotarsa decemlineata, CPB), or a Brazilian CornRootworm complex (BCR, consisting of Diabrotica viridula and Diabroticaspeciosa). Average low Root Damage Rating (RDR) scores of R₀ and F₁plants relative to non-transformed corn plant controls are demonstrativeof resistant to the corn rootworm species conferred by expression of thechimeric toxins.

All of the compositions disclosed and claimed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. While the compositions of this invention have been describedin terms of the foregoing illustrative embodiments, it will be apparentto those of skill in the art that variations, changes, modifications,and alterations may be applied to the compositions described herein,without departing from the true concept, spirit, and scope of theinvention. More specifically, it will be apparent that certain agentsthat are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope, andconcept of the invention as defined by the appended claims.

All publications and published patent documents cited in thespecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

What is claimed is:
 1. A recombinant nucleic acid molecule comprising aheterologous promoter operably linked to a polynucleotide segmentencoding a pesticidal protein or pesticidal fragment thereof, wherein:a. said pesticidal protein comprises the amino acid sequence of SEQ IDNO:119, SEQ ID NO:125, or SEQ ID NO:127; or b. said pesticidal proteincomprises an amino acid sequence having at least 95% identity to SEQ IDNO:119, SEQ ID NO:125, or SEQ ID NO:127.
 2. The recombinant nucleic acidmolecule of claim 1, wherein: a. the recombinant nucleic acid moleculecomprises a sequence that functions to express the pesticidal protein ina plant; or b. the recombinant nucleic acid molecule is expressed in aplant cell to produce a pesticidally effective amount of the pesticidalprotein; or c. the recombinant nucleic acid molecule is in operablelinkage with a vector, and said vector is selected from the groupconsisting of a plasmid, phagemid, bacmid, cosmid, and a bacterial oryeast artificial chromosome.
 3. The recombinant nucleic acid molecule ofclaim 1, comprised within a host cell, wherein said host cell isselected from the group consisting of a bacterial and a plant cell. 4.The recombinant nucleic acid molecule of claim 3, wherein the bacterialhost cell is from a genus of bacteria selected from the group consistingof: Agrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia,Pseudomonas, Klebsiella, Pantoea, and Erwinia.
 5. The recombinantnucleic acid molecule of claim 4, wherein the Bacillus species isBacillus cereus or Bacillus thuringiensis, said Brevibacillus isBrevibacillus laterosperus, or said Escherichia is Escherichia coli. 6.The recombinant nucleic acid molecule of claim 3, wherein said plantcell is a dicotyledonous or a monocotyledonous plant cell.
 7. Therecombinant nucleic acid molecule of claim 6, wherein said plant hostcell is selected from the group consisting of an alfalfa, banana,barley, bean, broccoli, cabbage, Brassica, carrot, cassava, castor,cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut, coffee,corn, clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant,eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblolly pine,millets, melons, nut, oat, olive, onion, ornamental, palm, pasturegrass, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin,Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub,sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato,switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon,and wheat plant cell.
 8. The recombinant nucleic acid molecule of claim1, wherein said pesticidal protein exhibits activity against aColeopteran insect.
 9. The recombinant nucleic acid molecule of claim 8,wherein said insect is Western Corn Rootworm, Southern Corn Rootworm,Northern Corn Rootworm, Mexican Corn Rootworm, Brazilian Corn Rootworm,Colorado Potato Beetle, Brazilian Corn Rootworm complex consisting ofDiabrotica viridula and Diabrotica speciosa, Crucifer Flea Beetle,Striped Flea Beetle, or Western Black Flea Beetle.
 10. The recombinantnucleic acid molecule of claim 1, wherein said pesticidal proteinexhibits activity against an insect species of the order of Lepidoptera.11. The recombinant nucleic acid molecule of claim 10, wherein saidinsect is Black Cutworm, Corn Earworm, Diamondback Moth, European CornBorer, Fall Armyworm, Southern Armyworm, Soybean Looper, SouthwesternCorn Borer, Tobacco Budworm, Velvetbean Caterpillar, Sugarcane Borer,Lesser Cornstalk Borer, Black Armyworm, Beet Armyworm, Old WorldBollworm, Oriental leaf Worm, or Pink Bollworm.
 12. A plant or partthereof comprising the recombinant nucleic acid molecule of claim
 1. 13.The plant or part thereof of claim 12, wherein said plant is a monocotplant or a dicot plant.
 14. The plant or part thereof of claim 12,wherein the plant is selected from the group consisting of an alfalfa,banana, barley, bean, broccoli, cabbage, Brassica, carrot, cassava,castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut,coffee, corn, clover, cotton, a cucurbit, cucumber, Douglas fir,eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblollypine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasturegrass, pea, peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin,Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub,sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato,switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon,and wheat.
 15. A seed of the plant of claim 12, wherein said seedcomprises said recombinant nucleic acid molecule.
 16. An insectinhibitory composition comprising the recombinant nucleic acid moleculeof claim
 1. 17. The insect inhibitory composition of claim 16, furthercomprising a nucleotide sequence encoding at least one other pesticidalagent that is different from said pesticidal protein.
 18. The insectinhibitory composition of claim 17, wherein said at least one otherpesticidal agent is selected from the group consisting of an insectinhibitory protein, an insect inhibitory dsRNA molecule, and anancillary protein.
 19. The insect inhibitory composition of claim 17,wherein said at least one other pesticidal agent exhibits activityagainst one or more pest species of the orders Lepidoptera, Coleoptera,or Hemiptera.
 20. The insect inhibitory composition of claim 19, whereinsaid at least one other pesticidal protein is selected from the groupconsisting of a Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1C,Cry1C variants, Cry1D, Cry1E, Cry1F, Cry1A/F chimeras, Cry1G, Cry1H,Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variants,Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35, Cry43A,Cry43B, Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TIC400, TIC407,TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901, TIC1201,TIC1415, TIC2160, TIC3131, TIC836, TIC860, TIC867, TIC869, TIC1100,VIP3A, VIP3B, VIP3Ab, AXMI-AXMI-, AXMI-88, AXMI-97, AXMI-102, AXMI-112,AXMI-117, AXMI-100, AXMI-115, AXMI-113, and AXMI-005, AXMI134, AXMI-150,AXMI-171, AXMI-184, AXMI-196, AXMI-204, AXMI-207, AXMI-209, AXMI-205,AXMI-218, AXMI-220, AXMI-221z, AXMI-222z, AXMI-223z, AXMI-224z andAXMI-225z, AXMI-238, AXMI-270, AXMI-279, AXMI-345, AXMI-335, AXMI-R1 andvariants thereof, IP3 and variants thereof, DIG-3, DIG-5, DIG-10,DIG-657 DIG-11, Cry71Aa1, Cry72Aa1, PHI-4 variants, PIP-72 variants,PIP-45 variants, PIP-64 variants, PIP-74 variants, PIP-75 variants,PIP-77 variants, Axmi422, Dig-305, Axmi440, PIP-47 variants, Axmi281,BT-009, BT-0012, BT-0013, BT-0023, BT0067, BT-0044, BT-0051, BT-0068,BT-0128, DIG-17, DIG-90, DIG-79, Cry1JP578V, Cry1JPS1, and Cry1JPS1P578V.
 21. The insect inhibitory composition of claim 16, comprisinga plant cell that expresses said recombinant nucleic acid molecule. 22.A commodity product produced from the plant or part thereof of claim 12,wherein the commodity product comprises a detectable amount of saidrecombinant nucleic acid molecule or a pesticidal protein encodedthereby.
 23. The commodity product of claim 22, selected from the groupconsisting of commodity corn bagged by a grain handler, corn flakes,corn cakes, corn flour, corn meal, corn syrup, corn oil, corn silage,corn starch, corn cereal, whole or processed cotton seed, cotton oil,lint, seeds and plant parts processed for feed or food, fiber, paper,biomasses, and fuel products such as fuel derived from cotton oil orpellets derived from cotton gin waste, whole or processed soybean seed,soybean oil, soybean protein, soybean meal, soybean flour, soybeanflakes, soybean bran, soybean milk, soybean cheese, soybean wine, animalfeed comprising soybean, paper comprising soybean, cream comprisingsoybean, soybean biomass, and fuel products produced using soybeanplants and soybean plant parts.
 24. A method of producing seedcomprising: a. planting at least a first seed according to claim 19; b.growing a plant from the seed; and c. harvesting seed from the plant,wherein said harvested seed comprises said recombinant nucleic acidmolecule.
 25. A plant resistant to insect infestation, wherein the cellsof said plant comprise the recombinant nucleic acid molecule of claim 1.26. A method for controlling a Coleopteran or Lepidopteran species pestor pest infestation, said method comprising: a. contacting the pest withan insecticidally effective amount of a pesticidal protein as set forthin SEQ ID NO:119, SEQ ID NO:125, or SEQ ID NO:127; or b. contacting thepest with an insecticidally effective amount of one or more pesticidalproteins comprising an amino acid sequence having at least 95% identityto SEQ ID NO:119, SEQ ID NO:125, or SEQ ID NO:127.